Note: Descriptions are shown in the official language in which they were submitted.
- ' GH-70087 CA 02234414 1998-06-03
cDNA CLONE HEOAD54 THAT ENCODES A HUMAN 7-TRANSMEMBRANE
RECEPTOR
This ~,~ 1;.. claims the benefit of U.S. Plu.~ l AYL~ No. 60/050,124, filed June 18, 1997.
s
FIELD OF INVENTION
This invention relates to newly i~ t;l';ed pol~ ;d~ i, poly~ li~s encoded by them
and to the use of such poly..-~ I~t;~l~s and polypepti~es, and to their p~ More
10 particularly, the poly..--- le~ 1es and polypeptides ofthe present h~ ion relate to the G-protein
coupled receptor family, };~eh~a~LGl referred to as HEOAD54. The invention also relates to
ilLil,it~ or a~il,va~ g the action of such polyn~cle~ ;~s and polypep~i~s.
BACKGROUND OF THE INVENTION
15 It is well e~ I that many . ' 'ly .~ lu~; are .. l;-l~i by
proteins pal~ in signal 1~ v~ay~ that involve G-proteins andlor second ...~
e.g., cAMP (Leflcowitz, Nature, 1991, 351:353-354). Hereinthese proteins are referredto as proteins
pa~ in ~dll~ . y;~ with G-proteins or PPG proteins. Some; I ' ofthese proteins include the
GPC lC~ , such as those for ~ ,IIGI~ agents and dopamine (1~ , B.K., et al., Proc. Natl
Acad. Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650-656; Bunzow, J.R, et
al., Nature, 1988, 336:783-787), G-pro~eins tl~.l~elvGs, effector proteins, e.g., pl~ C, adenyl
cyclase, and phosphodiesterase, and actuator pr~s, e.g., protein kinase A and protein kinase C
(Simon, M.I., et al., Science, 1991, 252:802-8)
For ~ " in one form of signal l~h..~.h.. ~ the effect of horrnone binding is activation of
25 the enzyme, ~ldLG cyclase, inside the cell. Enzyme a~LiVa~ )ll by lh ., .... ~.~ is f~ ,fl~ .t on the
presence ofthe ~ , GTP. GTP also ;"n"~. ,. ~ hormone binding. A G-protein connects the
hf nnf ne receptor to adenylate cyclase. G-protein was shown to ~ .- 1,;..~ GTP for bound GDP when
activated by â honnf)nf receptor. The GTP caIIying form then binds to activated adenylate cyclase.
Hydrolysis of GTP to GDP, ~l~ly~cd by the G-pratein itself, ~rns the G-protein to its basal, inactive
30 form. Thus, the G-protein serves a dual role, as an intellll~lidl~ that relays the signal from receptor to
effector, and as a clock that con~fols the duration ofthe signal.
. CA 02234414 1998-06-03
GH-70087
-
The .. -1." -.~ pro~in gene S~IA r, ---;ly of G-protein coupled ,~~ has been ~ .h ~.
as h~ving seven putative ~ m -1~ The domains are believed to ~ t
1.,.. ~.. ,1.. ~.-~. a-helices ~ ~1 by e~P~ r or cytoplasmic loops. G~ro~in coupled
înclude a wide range of' -'~B Ily active 1~.~ i, such as hr~rm~e~ viral, grow~ f~ctor and
J~i~"llUI~Ci~l ,.
G-pro1~in coupled 1. ~d ~ (~i,~,.~.~ known as 7TM l~l)tu~) have been ~h~ 1 as
including these seven ~,~. ~1 L~ rl ~ ~ stretches of about 20 to 30 amino acids, c~ at least
eigl~it li~ t Ly~ upLliic loops. The G-pratein i~unily of coupled receptors includes d~ .p~
l~tUI~ which bind to ~ ir drugs used for trea~ng p~y~.L~iic and ~ lo~ . Other
10 ~ .' of ~ IA ,ofthisfamilyinclude,butarenatlirnitedto,calcitonin,a~ ,.~,e-~lull- li~"
,~
cAMP, ad~ , muscarinic, a~ct~l~Lol;..~, sero~nin, histan~ine, thi~ nin, follicle ~1; ,.--l~l;-~
hf~rmf n~., opsins~ e~ Al~f li~l dilrel~ o"i gene-l~ ~hù lu~ , odorant, and ~ f~virus I~)tul~.
Most G-proteiin coupled I~ ~ have single c~ v~i cysteine residues in each ofthe first
two ~ r loops which form disulfide bonds th~ are believed to stabilize r.~ A1 protein
15 structure. The 7 1~ regions are *~ 1 as TMI, TM2, TM3, TM4, TM5, TM6, and
TM7. TM3 has been implicated in signal 1,; .Ic.l... 1;~
Ph~lJL~lyldi~,iandliipidation(rAI ~ ior~i.~,~ i)ofcysteiineresiduescan;..n.
signal L i,~C~h~ of some G-protein coupled I~tUI~. Most G-protein coupled Iv~~tul~ contain
pf)~al phc~l .. ,. yldliiùI~i sites within thie third ~ia~ ' - loop and/or the carboxy t~rmin~c For
20 several G-protein coupled receptors, such as the ,B-a~ ,tu,, p~ lJllf)~ by protein kinase A
and/or specific receptor kinases mediates receptor ~l~c~ ;I;"n;,~
For some I~lJtw~, the ligand binding sites of G-protein coupled ~)tUl~ are believed to
c~ e Lywiu~ lic sockets formed by several G-protein coupled receptor l.,~ .. I.".. F d~mAi
said sockets being ~wiluull~xl by Lywi~h~ - residues ofthe G-protein coupled ~lJtvl~. The
25 hydrophilic side of each G-protein coupled receptor ~ I,- ,---- helix is poshll-~-d to face inward
and form a polar ligand binding site. TM3 has been ;~ Jli. ~ in several G-protein coupled l~/tul~ as
_aving a ligand binding site, such as the TM3 ~ te residue. TM5 serines, a TM6 ~ and
TM6 or TM7 pL~l~y~ I ~ or ly,u~l~ are also implicated in ligand binding.
-G-protein coupled l~G~tOl~ can be ;~ cel~ Iy coupled by hc~l~. ;.... ~ ;c G-proteins to
30 various intracellular enzymes, ion channels and L-~l.o.lc ~ (see, Johnson et al., Endoc. Rev., 1989,
10:317-331) Different G-protein a-subunits ~-~rt;,~ially ~ particular effectors to m~
va ious ' -'7.G 1 r.. ~;0.. ~ in a cell. Phu~l)L.~ of.;yhrl - - residues of G-protein coupled
CA 02234414 1998-06-03
GH-70087
,
ahavebeeni~ iasani..4~ul~...F ~ forthel~,.~ ,--ofG-proteincouplingof
some G-prote,in coupled l~l~tul~. G-protein coupled l~e~lS are found in lllJlll~ U:~ sites within a
. host.
Overthepast l5 years, nearly 350 ~ age~s ~ ,,.h.y~ 7 l~ F.
S l~wl~ have been j..,~,~.~r..lly i~c~l into the mar~et.
~ This '' -~ that these l~la have an ~l~hl;~l FA, proven history as 1}. ~ ;c tar~s.
Clearly there is a need for ~ 1; rr~ and cl ~ ;o~ of further l~: -r which can play a role in
pl~ "~ or c~ r~ or diseases, - -' '' ~, but not limited to, ;..r~
such as b~ , f~ ~ o~ and v~ fi ~ , p~uLly;~f~ l;ol~C caused by HIV-l or HIV-
10 2; pain; cancers; an~ ,~; bulimia; asthma; Palk~'a disease; acute heart failure; L~h. -: ~n;
Ly~ tl,~lùl~; urinary I~Li~7 Oa~V~Iua;a, angina pectoris; myoca~dial illLu-liull, ulcers; asthma;
~" -, , benign prostatic Ly~ ùlJlly, and pay~,Lvtic and I~ lo,~ a, including anxiety,
5~ ul a7 manic ~lcaa;ull7 delirium7 tlP~nPnti~, severe mental Ict~udaiùll and dy~ c, such as
TT~ 'a disease or Gilles dela Tourett's .7yll~hui~e.
SUMMARY OF THE INVENTION
Tn one aspect7 the invention relates to HEOAD54 pOl~ a and ~c~ .1 materials and
methods fortheir plUll"~ Another aspect ofthe ilI~ ltiuIl relates to methods for using such
HEOAD54 poly~G~ti L7 and poly~ f s Such uses include the ~ lult of in~rti-nc such as
20 ~P.~1, fun~l, I,l~O~ and viral ;..1~ , particularly ;..r~;m-~ caused by H[IV-l or H[IV-2; pain;
cancers; anon~xia7 bulimia7 asthma; Pcul~laai'~ disease; acute heart failure; Ly~t ~ - h-y~clb~L7;ull~
urinary I~t~tiU~ ~7l~V~Iu.7;a, angina pectoris; lllyo~icud;al illLu~liùl~; ulcers; asthma; ~l1F~1F~C; benign
prostatic LYI~G1 llu~Ly; and pay-,Lùtic and neul~ ir~ ela, including anxiety, s~;1.;,I-~J1~ f~a~ manic
de~JI~a;ùll~ ~ldirjllrn, ~1~n~ti~, severe mental I~lcu~L~tivl~ and ~ c, such as IT ~~ ,t~ s disecase
25 or Gilles dela Tourett's syndrome, among others. Tn still another aspect, the invention relates to
methods to identify agonists and ~nt~ ni~tc using the m~t~ provided by the invention, and
treating co~ .c ~c~o~ ~ with ~OAD54 imh~l~nt~ with the i.l~ .. "; rf~l Cvlll~JUui~l.ki. Yet another
aspect of the ,ll~ tiu.. relates to ~ ;r assays for ~ diseases - ~ with il~a~)~l~l;~
HEOAD54 activity or levels.
GEI-70087 CA 02234414 1998-06-03
DESCRIPTION OF T~ INVENTION
Defini~ons
The following ~r;~ ;""c are provided to f~rilit~te ull~cl~ n~ g of certain terms used
lic4u~ 1y herein.
"HEOAD54" refers, among others, to a poly~ tide ~ P the amino acid seqluPnre
set forth in SEQ ID NO:2, or an allelic variant thereof.
"Receptor Activity" or "Rislogr~l Activity ofthe Receptor" refers to the metabolic or
physiologic fimr,tirm of said HEOAD54 inr111rling similar a~,Liviti~,s or illl~,lo~l activities or these
a~livi~s with d~,~s~ ~ '~ side-effects. Also inr~ P~ are ~ ;r and ;~ -.Ogf ~;c
~liviLics of said HEOAD54.
"HEOAD54 gene" refers to a pOlynllrl~Qti~eculll~ ~g the ~1~rleotiflP sequ-pnce set forth
in SEQ ID NO: l or allelic variants thereof and/or their uj"~ npntc .
".AntihofliP,s" as used herein includes polyclonal and .... I~oclc~ antibodies"1~;...- ;~,
single chain, and 1----. ~ ntibor1ips~ as well as Fab r,;.~..l..n~ fl...l;.~g the pl~nluct~ of an Fab
15 or other ;~ oglobulin tA~ ;O~ library.
'Ysolated" means altered "by the hand of man" from the natural state. If an "isolated"
wlll~oa;tion or ~Ub~ .Ce occurs in nature, it has been changed or removed from its original
cnvilulllll~ t~ or both. For ",, _ r1f, a polym~rlPotirl-P or a polypeptide naturally present in a living
animal is not "isolated," but the same polym~rlP,oti(lv or poly~clltide sc~dlated from the coc~ ;~l ;u~
20 m~tPri~lc of its natural state is "isolated", as the term is c~ luycd herein."Polyn--rleoti~l-P~ generally refers to any poly,;b-l. .- ~IP~I ;~1P or polydeuA-;bo~ . -rlPotir1P,
which may be ..I....~l;I;ed RNA or DNA or m~ified RNA or DNA. "Polym~lP~ f-s" include,
without 1;...; ~ ;.... single- and doul~'o str~n-lPd DNA, DNA that is a mixture of single- and double-
~nrlP,d regions, single- and double-str~n~lp~d RNA, and RNA that is mixture of single- and
25 double-stranded regions, hybrid 1 I-PS co,ll~ ing DNA and RNA that may be single-stranded
or, more typically, dol.~le-~al~ded or a mixture of single- and double-stranded regions. In
a~l-liti- n ''polym~rlPiot~ refers to triple-stranded regions COlll~ g RNA or DNA or both RNA
and DNA. The term polym~r1PQti~1p also includes DNAs or RNAs c~ g one or more m~ified
bases and DNAs or RNAs with ba~ L1"~n-Ps ...~I;I~PA for stability or for other reasons. "Modified"
- 30 bases include, for ~ rl~ ;LylaLcd bases and unusual bases such as inosine. A variety of
mrxlifir~tir,n~ has been made to DNA and RNA; thus, "polyn~c1eoti~l-P-" embraces chpmic~lly7
enzymatically or m-Pt~br~lir~lly ~ 1; r;ed forms of polyn~r1eoti-les as typically found in nature, as
' CA 02234414 1998-06-03
GEI-70087
.
well as the chcmical forms of DNA and RNA cl~ua~ ;c of viruses and cells. "PolrllrlPoti~lP"
also cmbraces ,~,lalively short polynl~rleoti~lPc~ o~en referrcd to as oli~nllrlP,otides.
"Polypcptide" refers to any peptide or protein C~ ,isi lg two or more amino acids joincd
to cach other by peptide bonds or ,..r ~ P,d peptide bonds, i.e., peptide iso~les. "Polypcptide"
5 refers to both short chains, ~ ly referred to as pepti~Pc~ nlig..~Jt;flPS or oligr,mPrs, and to
longer chains, generally referred to as proteins. Poly~ ,tid~s may contain amino acids other than
the 20 ~,e.~ odc~ amino acids. "Poly~lides" include amino acid s~ ,es ".n l;l;ed either by
natural pnx~;,~es, such as po~ l;r,n~l pr~ec~ , or by chpmir~l mly1ifir,~ti~m tP~rhni~lnps
which are well known in the art. Such m~ifir~tionc are well describcd in basic texts and in more
10 detailed ,llono~a?hs, as well as in a vol ~",;~,O~C~ rescarch li~ u~ lo lifie~ti. nc can occur
~y~hcl~; in a polyl.c~tide, inr.lllrli~ the peptide backbone, the amino acid side-chains and the
amino or C~bOAYI tem~ini. It will be a~ ,;ated that the same type of, r~ f ;- ..~ may be
present in the same or varying degrees at several sites in a given polypeptide. Also, a given
polypeptide may contain many types of mr~ifir~tirlnc2 Poly~lides may be blanch~ as a result of
~b ~ l ;r~n and they may be cyclic, with or without b. ~ ;~ Cyclic, blancl1ed and b,ail~,llcd
cyclic polypeptides may result from po~ natural plocesses or may be made by synthetic
mPth-u1c. l~o~ifir~ti~nc include ac~ylalion, acylation, ADP-ribosylation, ~mi-l~tion, covalent
att~rhm-Pnt of flavin, covalent ~tt~rhmPnt of a heme moiety, covalent ~tt~.h~ 1 of a mlrlP~oti~P or
n~lrlPotide derivative, covalent ~tt~rhm~nt of a lipid or lipid derivative, covalent ~tt~rhmPnt of
phf~5~h~ lyl;l~0~ l1 cross-linking, cycli7~tirln fliclllfi~r bond form~tirJn d~ -ylalion, ro~ l;r~l-
of covalent cross-links, fiorm~tion of cystine, fiorm~tion of py-ùgl~ le, formylation, gamma-
u~ylation, glycosylation, GPI anchor fonn~tirn, hydlu~ylalion~ io lin~tinn, Illc;lhylalion,
myristoylation, o~ tinn~ proteolytic p~uces~ing, pho~hn~ylalion~ prenylation, racr.mi7~tinn~
sele,.uylalion~ slllf~fir~n, transfer-RNA ~--~ trd addition of amino acids to proteins such as
~ laLion, and ubi~ l ;u~. See, for inct~nrx, PROTElNS - STRUCTURE AND
MOLECULARPROPERl~S,2ndEd.,T. E. Creighton,W. H. F,ee.~ andCompany,New
York, 1993 and Wold, F., po-,ll~ l Protein ~ifir.~tionc Perspectives and P~u.,~e~
pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., ~r.~ .mir, Press, New York, 1983; Seifter et aL, "Analysis for protein m~ifir.~ti( ns
and nulllJlul~h~ cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein
Synthesis: po~ ti~n~l Mo lifir~tirmc and Aging", Ann NYAcad Sci (1992) 663:48-62.
GH-70087 CA 02234414 1998-06-03
"Variant" as the term is used herein, is a polym~cl~P,otide or polypeptide that differs from a
.crcl~,ncc polyn~r1eQt~ P or poly~ tide lC'~lf ,_Li~,~,ly, but retains çccPnti~1 plup~ ,s. A typical
variant of a polyn--~lP~ticle differs in n~1r1P~tif~P se~ ,e from another, lcrc~ cc polynuc1Pvoti~l-P-
Changes in the ~u~1P~1;de s~ e of the variant may or may not alter the amino acid 5~1~ e of
5 a pol~.,~Li~ encoded by the ~cÇcl~ ce pol~..--~1e~ 1c Nv~1Pvoti~lP changes may result in amino
acid ,~ liti~nc, ~If~ , fusions and tr~m~ ti~ ns in the poly~c~ti le encoded by the
l~,f~ ,c s~ c~" as ~ , c~ below. A typical variant of a polypeptide differs in amino acid
se~Pnee from another, Icrcl-,~lcc polypeptide. Generally, dirrtl-,nces are limited so that the
S~u ~'f ,S of the If f~ lce polypeptide and the variant are closely similar overall and, in many
10 regions, i-lPntit~1 A variant and lcîcl~,ncf polypeptide may differ in amino acid sequpnre by one or
more ,-~h~ 1l;o~c, ~liti~nci"lPletion~ in~ any colll~illd~ion. A ~ub~ 1f~ or inserted amino acid
residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or
poly~ )Lide may be a naturally OCCulllllg such as an allelic variant, or it may be a variant that is
not known to occur naturally. Non-naturally oc~ variants of polynuclcutides and
15 polypeptides may be made by --1~r-.- ~;c tP~hniTles or by direct syllLll.,sis.
'Ydentity" is a measure of the identity of nllrle~ti~lP se~u '~f ~' or amino acid sequpn~s. In
general, the s~ ,s are aligned so that the highest order match is obt~in-p~ "Identity" per se has
an art-lc~o~ G and can be c~ ted using published ~ e~ See, e.g.:
(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford Ullivcl~iLy Press, NewYork, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W.,
ed., ~'ZU~f'miC Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA,
PART I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; SEQUENCE
ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G.,1'7e7~1rmir. Press, 1987; and
SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). While there exist a number of methods to measure identity between two
polylPlclf,otide or poly~,c;ytide se~uenr~C~ the term "identity" is well known to skilled artisans
(Carillo, H., and Lipton, D., SL9M JApplied Math (1988) 48: 1073). Methods comm~7nly
,loyed to ~ f identity or similarity between two sf~uenrf,s include, but are not limited to,
those ~l;C~lose~ in Guide to Huge Coll~ul~l~, Martin J. Bishop, ed., .A-~,7.-1rmic Press, San Diego,
1994, and Carillo, H., and Lipton, D., SLAM JApplied Math (1988) 48: 1073. Methods to
d~ identity and similarity are codified in c~ . progl~..s. Pl~llc;d C(Jlll~JUkil pil~l~lll
methods to dt~tr~ " ~;~,r identity and similarity between two sequrnces include, but are not limited to,
. . .
.. . . .. , ~ .. ., , .. .. . .. . -, ... . .. -, - . : .. . .... . ..
GEI-70087 CA 02234414 1998-06-03
GCS ylO~;Ialll package (Devereux, J., etal., NucleicAcids Research (1984) 12(1):387), BLASTP,
BLASTN, FASTA (Atschul, S.F. et al., JMolec Biol (1990) 215:403).
As an i~ ; l ;on, by a polym~rleot~ o having a ~,..rlo~l ;.1o s~u~-n~e having at least, for
, 1, 95% "identity" to a l~,f~ lce mlr,leotir~ se~ nr~ of SEQ ID NO: 1 is intended that the
5 ~ le~l ;fio se~u ~re of the pol.~ rle~ is identical to the l~fel~ ce seql~onre except that the
yOl~ 1PU~ nr~ may include up to five point n-llt~ti- nc per each 100 mlrleQtitl-o~s of the
Icr~ "~ n ~l~n;~ s~lu---~-~ of SEQ ID NO: 1. In other words, to obtain a polymlrlo,otitl-o~ having
a mlrlooti~o s~lu- ~~eG at least 95% i-~ontir~l to a l~rtil-,.lce nllrleotirlo s~.,- .-~, up to 5% of the
mlrleotitlos in the l.,rel~ncP seq~lonr~e may be deleted or s~,b ,I;l~ 1 with another mlrlo,oti~o7 or a
10 number of ""~1P~I ;-lf c up to 5% of the total ml~ es in the It;rc~ ,C s~u- ~-,e may be inserted
into the l~rt;l.,llce seqU-once These mllt~tionc of the Itir~l-,.lce sequ-onr,e may occur at the 5 or 3
terminal positions ofthe l~r~ ce nllcleot~ o s~lu- ~--~ or allywl~.~i between those terminal
pocitirnc, illtt;l sye-:~ed either individually among ..-.rl~ es in the Ic;r~lcllce sequeonre or in one or
more co~ .1 ;g, -~- ~c groups within the l~relcnc s~u- . .~.
Similarly, by a polyy~ tide having an amino acid se~l.,- ~.-,e having at least, for ~llyle~
95% "identity" to a l~irel~"lce amino acid se~ r~ of SEQ ID NO:2 is inPntled that the amino acid
sequenre of the polypeptide is i-lontir~l to the lerel~,nce sequonre except that the polypeptide
s~ e may include up to five amino acid alterations per each 100 amino acids ofthe lcrc-cllce
amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid
20 se~u-onre at least 95% i-lon*c~l to a lcrc--,llce amino acid se~lu- ~re, up to 5% of the amino acid
residues in the ~crc-cll~e se~ l-onre may be deleted or sub~.l;l~llet1 with another amino acid, or a
number of amino acids up to 5% of the total amino acid residues in the lcrcl~ ce s~ e may be
inserted into the lcrclGncc seql~-onre. These al~clalions ofthe Icrclcl~e sequ-o~re may occur at the
amino or carboxy terminal po~i*rlnc of the lcrcl~,nce amino acid sequ-onr,e or cul~wh~c between
25 those terminal positions, ;~ yel~7cd either individually among residues in the .crc-c.lce seqU~nce
or in one or more conti~ groups within the lcrw~.lce sequence.
Pol~ Jtid~. of the In~rention
In one aspect, the present invention relates to HEOAD54 yOl~ycy1idc;7 (or HEOAD54
30 proteins). The HEOAD54 polypeptides include the polypeptides of SEQ ID NOS:2 and 4; as well as
polypeptides culllyli~ g the amino acid seqU~nre of SEQ ID NO:2; and polypeptides colllylisillg
the amino acid s~u~nce which have at least 80% identity to that of SEQ ID NO:2 over its entire
Gll-70087 CA 02234414 1998-06-03
.
length, and still more preferably at least 90% identity, and even still more preferably at least 95%
identity to SEQ ID NO: 2. Fu lL~ Ie, those with at least 97-99% are highly ~lIGrG~lGd. Also
included within HEOAD54 polypeptides are poly~ tides having the amino acid seq~l~nce which
have at least 80% identity to the polypeptide having the amino acid sequPnre of SEQ ID NO: 2
5 over its entire length, and still more preferably at least 90% identity, and even still more preferably
at least 95% identity to SEQ ID NO: 2. Fu.lL~.,.orG, those with at least 97-99% are highly
p.~,fe..Gd. P-~rc ably, HEOAD54 poly~ )tides exhibit at least one biolcO ~.~l activity ofthe
receptor.
The HEOAD54 poly~ Jtid~s may be in the form of the "mature" protein or may be a part
10 of a larger protein such as a fusion protein. It is often adv~nt~g~o~lc to include an ~ liti~n~l amino
acid sc~ cc which contains S~lGt~ ly or leader se~ ,s, pro-s~.l~ PC, se lu~ - .CGS which aid in
,u~;r~l;cn such as multiple l-: d;.l;.,e residues, or an ,q~ ;o~l se4~lr~'e for stability during
~w)n~ a~1 pro~ ion
F.~..- .~t~ ofthe HEOAD54 polypeptides are also included in the ill~ ~,.ltiol.. A r.~.,. .,1 is a
15 p. I.y~ytidG having an amino acid ~1u .- f tbat entirely is the same as part, but not all, ofthe amino acid
~ u~.~ e ofthe ar~ ;o~ HEOAD54 poly~ti~. As withE~OAD54 pol~ ti~ " r.~,.... ,
may be "free standing," or c~ ~I" ;~1 wi~in a larger pol~l/G~ti IG of which they form a part or region,
mostpl~,f~.ably as a single ~ r~Y region. RGl"~,~.~livG ,~ , 1 of poly~JG~)tillG r.~..- ,1Y ofthe
ti~, include, for ~ r'e, fi~ t~ from about amino acid number 1-20, 2140, 41~0, 61-80,
81-100, and 101 to the end of HEOAD54 polypG~ti~G. Jn this context "about" includes the pal~i~;uldlly
recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both
plcrcllcd r ~--- -n~ include, for; , '-, llu~ poly~c~i~ having the amino acid
SC~Iu ~e of HEOAD54 polypeptides, except for deletion of a c~.. n; . . ~0~ ~C series of residues tbat includes
25 the amino terminus, or a ci ~-d;--~ series of residues tbat includes tbe carboxyl terminus or delction of
two ci ~- n ;~ y series of residues, one including the amino terminus and one including the carboxyl
AlsolJlcrc:llcdarer~ d~ A~A~ by!illu~ orr~ AlA1l~ ~suchas
r~ .. dt~ tbat c~ , alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-formulg
regions, turn and turn-forming regions, coil and coil-forming regions, hy~llo~ ilic regions, hydl~ b;-
30 regions, alpha ll ' ~r a~hic regrions, beta ~ , '~i, ~thic regions, fiexible regions, surface-forming regiorls,
substrate binding region, and high A- n ;~,r- ~ G index regions. Other p-crC.-~ fi A,~ ; are ~ ly
active r. ~ ,t~ P*l~ Ily active r. i~" . dt'; are those that mediate receptor activity, inrl~ in~ those
GEI-70087 CA 02234414 1998-06-03
with a simtlar activity or an il.~lO . ~i activtty, or with a d~~ activity. Also included
are those that are antigentc or ~" in an a~ especially in a human.
Pl~f~,.~ly, all ofthese pol~ti~ r.~. ~ retainthe b -Ic~ activity ofthe rece~tor,mcluding antigenic activity. Among the most ~ tÇ~ ~~AI r.~ 4 is thathaving the ammo acid 5~ e
5 of SEQ ID NO: 4. Variants ofthe defined S~ r ~ and r..~.. .,t~ also form part ofthe present
ill~i~. Pl~f~,.-~ -variants are those that vaIy from the rel;erents by Cu~ va~iv~ amino acid
,..I .,I;~. .I ;o. .~ - i.e., those that ~ a residue with another of like cl~ Typical such
~. .1 .~l ;1. Il ;n. .~ are among Ala, Val, Leu and ne; among Ser and Thr; among the acidic residues Asp and
Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
10 P~~ l~ly yl~f~, l~ are variants m which sevelal, 5-10, 1-5, or 1-2 amino acids are ,.lb'l;1
deleted, or added in any . ' ~
The HEOAD54 pol~ ofthe ill~ ll can be ~ in any suitable manner. Such
poly~)tid~; include isolated nalu~ y o~ g pol.~ ;J~, r~r~ 1y plulhl~d poly~.LiJe:3,
~"1l~t;~lly~ uc~lpol~ es~ orpoly~ l~lbyac~,..-l.~.~I;.~nofthese m~h~
15 Means for ~ g such poly~l ide~ are well ~ 'J,~od in the art.
Pobn ' ~'- ofthel~
Another a~pect ofthe illv~liUll relates to HEOAD54 pd),. ~ HEOAD54
pci~ f ~ include isolated poly- .- ~, 1 ~1 ;,l, ~ which encode the HEOAD54 poly~tid~ and
20 r. ~ . .~ , and pol~ )t;~lf~ closely related thereto. More specific~lly, HEOAD54 polynllcl~oti~s
of the illv~ n include a pol~ the ~ d in SEQ ID NO: le~ an HEOAD54 poly~ e of SEQ ID NO: 2, and poly. . ,- 1 ~n ;.1.~.~ having the p~ Li~,ula
f ~ of SEQ ID NOS: 1 and 3. HEOAD54 polynllrlP~ti~es further include a poly., ~r,l~.~JI ;tl~
a mlrlf!oti(l~ s~l-lr~ that has at least 80% identity over its entire length to a mlcle~ti~l~
25 se~u~nr~ c -r~l;ng the HEOAD54 polypeptide of SEQ ID NO:2, and a polynllrleotille comprising a
nllrleotirle s~lu~ e that is at least 80% irlentir~l to that of SEQ ID NO: 1 over its entire length. In
this regard, pol~..--- 11F~;~ at least 90% identical are particularly p,~r~ll~, and those with at least 95%
are especially ~ f~,.lcd. Ful~ l--ul~, those with at least 97% are highly pl~rell~d and those with at
least 98-99% are most highly pl~,f~ l, with at least 99% being the most plcr~ d. Also inrlu~le~
30 under HEOAD54 poly.~ t;~les are a mlrleoti~le se~u~nr~ which has s~lffici~nt identity to a
mlrlPotitl~ se~uenr,e c~ ;..~ in SEQ ID NO:l to Lybli~lize under cQn~liti~n~ useable for
: 9
.
GH-70087 CA 02234414 1998-06-03
n or for use as a probe or marker. The i~ tion also provides polyml~ des which
are Cu~ to such HEOAD54 polynllclP~es.
HEOAD54 of the ill-~iUll iS ~11 U ~ ly rela~ed to other proteios of the G-prot_in coupled
receptor fi~mily, as shown by the results of ~~ the cDNA encoding hurnan HEOAD54. The
cDNA ~I~ f, of SEQ lD NO:l contaios an open reading frame (,.. ~I. 4;.l~ ournber 236 to 1504)
._c " ,, a ~I~ of 423 amino acids of SEQ ID NO:2. The amino acid 5~1~ e of Table 1 (SEQ
lD NO:2) has about 40.2 % ident~ (usiog FASTA) in 291 amino acid residue~s with human l,lol 3}1e G
pr~in coupled receptor, HM74 (AC~ ;~)n # P49019, Nomula, H. et al, lnt. Tmmlml~l 5: 1239-1249,
1993). Fu~ , HEOAD54 (SEQ lD NO: 2) is 27.3 % identical to human P2U ~ over
341 amioo acid r~sidues (Ar~Q;~n # P41231, Parr, C.E. et al, Proc. Natl. Sci. U.S.A. 91: 3275-3279,
1994). The ~ r ~ of Table 1 (SEQ lD NO: 1) has about 98.25 % iden*ty (usiog BL,AST)
in 171 I,, ,~ lr~ residues with yc63gO5.rl Homo sapiens cDNA clone 85400 5' (Acc~cQ;~ n # T72122,
Wilson, R et al, Wa~shU-Merck EST project, Unr ' ' ih~ 1995). Ful IllCllllUlt;, HEOAD54 (SEQ lD
NO: 1) is 55.59 % identical to human G-protein coupled receptor mRNA over 349 nucleic acid residues
(Ar~Q;~n # U35398, An,S. et al, U ~ h~h~ 1995). Thus, HEOAD54 ~1~1id~ and
pol~ . .- -- 1~ ;,1~ of the pres~t invention are expected to h~ave, inter alia, similar ' ICBir
r.. ~ ;U. ~Iu~.~i~ to their 1~ )tiJ~ and pûl~ ec~ and their utili~r is obvious to
- an~one skilled in ~e art.
<
Table la
1 GACTATCCTC C QCTTCAGG GlLl~l~lGG GCTTCCATCT TGCCCCTGCT
51 GAGCCCTGCT lC~lC~l~lA CCAGCAGCAC AACCCCCAGG CTGGGCTCAG
101 AGACCTCATG TGGTGGGATC ACTCAGTACC CCGAGGCGGA GGGAAGGAGG
151 GAGGGCTGCA GG~LlCCC~l TGGCCTGCAA ACAGGAACAC AGG~lGlllC
201 TCAGTGGCTG CGAGAATGCT GATGA~AACC CCAGGATGTT GTGTCACCGT
251 GGTGGCCAGC TGATAGTGCC AATCATCCCA CTTTGCCCTG AGCACTCCTG
301 CAGGGGTAGA AGACTCCAGA AC~1L~1C'1C AGGCCCATGG CCCAAGCAGC
351 CCATGGAACT TCATAACCTG AG~l~lCCAT CTCCCTCTCT CTC~lC~
,' 10
' GEI-70087 CA 02234414 1998-06-03
401 ~Ll~lCC~lC C~lC~ll~lC lCC~l~ACCC lC~l~lGCTC CCTCTGCCTT
451 TACCACTGTG GGGGGGTCCT CTGGAGGGCC CTGCCACCCC AC~1~11CCT
501 CG~1G~1C l~C~l~C~lG GCACCAATCC TGGCC~1GGA ~111~1C~1G
551 GGC~1~1GG GGAACAGTTT GGCC~1~1 lC A1~L1~1GCA TCCACACGCG
601 GCCCTGGACC TCCAACACGG I~11C~1~L CAGC~1G~1G GCCGCTGACT
651 lC~lC~l GAT CAGCAACCTG CCC~LCCGCG TGGACTACTA CCTCCTCCAT
701 GAGACCTGGC G~lLlGGGGC TGCTGCCTGC AAAGTCAACC TCTTCATGCT
751 GTCCACCAAC CGCACGGCCA GC~11~11 CCTCACAGCC ATCGCACTCA
801 ACCGCTACCT GAAGGTGGTG CAGCCCCACC ACGTGCTGAG CCGTGCTTCC
851 GTGGGGGCAG ~1aCCCGG~L GGCCGGGG~A CTCTGGGTGG GCATCCTGCT
901 CCTCAACGGG CACCTGCTCC TGAGCACCTT CTCCGGCCCC TCCTGCCTCA
951 GCTACAGGGT GGGCACGAAG CC~1CGGCCT CGCTCCGCTG GCACCAGGCA
1001 CTGTACCTGC TGGAGTTCTT CCTGCCACTG GCGCTCATCC TCTTTGCTAT
1051 TGTGAGCATT GGGCTCACCA TCCGGAACCG 1G~1~1~GGC GGGCAGGCAG
1101 GCCCGCAGAG GGCCATGCGT GTGCTGGCCA TGGTGGTGGC CGTCTACACC
1151 ATCTGCTTCT TGCCCAGCAT CA1~1L1GGC ATGGCTTCCA TGGTGGCTTT
1201 CTGGCTGTCC GCCTGCCGCT CCCTGGACCT CTGCACACAG CTCTTCCATG
1251 GCTCCCTGGC CTTCACCTAC CTCAACAGTG TCCTGGACCC CGTGCTCTAC
a 1301 TG~.1~1~ l'A GCCCCAACTT CCTCCACCAG AGCCGGGCCT TGCTGGGCCT
1351 CACGCGGGGC CGGCAGGGCC CAGTGAGCGA CGAGAGCTCC TACCAACCCT
1401 CCAGGCAGTG GCGCTACCGG GAGGCCTCTA GGAAGGCGGA GGCCATAGGG
G~I-70087 CA 02234414 1998-06-03
.
1451 AAGCTGAAAG TGCAGGGCGA G~~ G GA~AAGGAAG G~C~.~CCA
1SO1 ~G~l~AGGG CCAGCTGCAG GGCTGCAGCG ~ ~GGG~ AAGGGCTGCC
1551 GCG~l-LGGC CTGGAGGGAC AAGGCCAGCA CACGGTGCCT CAAC
A nucleotide sequence of a human HEOAD54 (SEQ ID NO: 1).
Table 2-
1 MLCHRGGQLI VPIIPLCPEH S'~T~ R~T~QNT LSGPWPKQPM ELHNLSSPSP
-, 51 SLSSSVLPPS FSPSPSSAPS AFTTVGGSSG GPCHPTSSSL VSAFLAPILA
101 LEFVLGLVGN SLALFIFCIH TR~nl~N~v~ LVSLVAADFL LISNLPLRVD
151 YYLLHETWRF GAAACKVNLF MLSTNRTASV VFLTAIALNR YLK-vVQPHHV
201 LSRASVGAAA RVAGGLWVGI LLLNGHLLLS TFSGPSCLSY RVGTKPSASL
251 RWHQALYLLE FFLPLALILF AIVSIGLTIR NRGLGGQAGP QRAMRVLAMV
301 VAVYTICFLP SIIFGM~SMV AFWLSACRSL DLCTQLFHGS LAFTYLNSVL
351 DPVLYCFSSP NFLHQSRALL GLTRGRQGPV SDESSYQPSR QWRYREASRK
401 AEAIGKLKVQ GEVSLEKEGS SQG
~ An amino acid sequence of a human HEOAD54 (SEQ ID NO: 2).
One pol)/.~ ofthe present ~ ' 6 HEOAD54 may be obtained using
standard cloning and SCl~ ~ g, frorn a cDNA library derived from mRNA in cells of human ~lyitlr~Fhilc
using.the el~/lC:~S~l se~ tag (EST) analysis (Adams, M.D., et al. Science (1991) 252: 1651-
1656; Af~ns, M.D. et al., Nature, (1992) 355:632-634; Arlams, M.D., et al., Nature (1995) 377
Supp:3-174). Poly.-~r~ ;rlec ofthe invention can also be obtained from natural sources such as
genomic DNA libraries or can be syntheci7ed using well known and crJllu~lclcially available
, .-
es
The mlrl~tirl~ se1venre e- r~l;l~e the HEOAD54 poly~ Jtide of SEQ ID NO:2 may be
15 i-l~tir~l to the poly~c~ e ~-~ro li~ seq~nre co--~ r~1 in Table 1 (~ rl~ r~ number 236 to 1504
GEI-70~87 CA 02234414 1998-06-03
of SEQ ID NO: 1), or it may be a se~ , which as a result of the I~J ~ n~ ~ (deg~,.~.a~,y) of the
genetic code, also encodes the poly~ tide of SEQ ID NO:2.
When the pol~ s of the invention are used for the l~,~,Ulllbillalll, prorlllcti~n of the
HEOAD54 polypeptide, the polymlcleoti~e rnay include the coding ~ for the mature
5 polypeptide or a r."~ thereof, by itself; the coding s~ , forthe rnalure polypeptide or r.~...-."
in reading frarne with other coding ~l~ ~ such as those ~ ~ 1 g a leader or s~lduly s~ " a
pre, or pro- or prepro- protein ~ ., or other fusion peptide portions. For c , '~, a marker
~IU - ~ ~ which facilitates Ir~ ofthe fused pol~ ~ can be encoded. In certain ~
embodimerlts ofthis aspect ofthe ill~iUl~ the marker ~l~ ~ is a hexa-histidine peptide, as provided
inthe pQE vector (Qiagen, Inc.) and .1~.,. . il~1 in Gentz etaL, ProcNatl Acad Sci USA (1989) 86:821-
824, or is an E~A tag. The pol~ e may also coIItain non coding 5' and 3' s~ ~, such as
non~ d ~Il r-~--~7 splicing and pol~a~cllylaliull signals, . ;lY.~.".~ binding sites and
r~ a,t stabilize mRNA.
Further ~lcrcllcd embodiments are poly.~ , HEOAD54 variants c~
15 the arnino acid s~ f - ~e of the HEOAD54 pG~ ic of Table 2 (SEQ ID NO:2) in which several,
5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are .,~b.,l;l~ 1 deleted or added, in any e
Arnang~e l~lcrcllcd p~ 1 4;~1f s of ~e present ill~ is c....~ in Table 3 (SEQ ID NO: 3)
encoding~e amino acid sequence of Table 4 (SEQ ID NO: 4).
Table 3c
GGCACGAGCTGCCTTCCTGGCACCAATCCTGGCCCTGGAGTTTGTCCTGGGCCTGGTGGGGAACAGTTT
GGCCCTCTTCATCTTCTGCATCCACACGCGGCCCTGGACCTCCAACACGGTGTTCCTGGTCAGCCTGGT
GGCCGCTGACTTCCTCCTGATCAGCAACCTGCCCCTCCGCGTGGACTACTACCTCCTCCATGAGACCTG
GCGCTTTGGGGCTGCTGCCTGCAAAGTCAACCTCTTCATGCTGTCNACCAACCGCAAGGCCAGCGTTGT
CTTCCTCACAGCCATCGCACTCAACCGCTACCTGAAGGTGGTGCANCCCCACCACGTGCTGAACCGTGC
TTCCGTGGGGGCANCTGCCCGGGTGGNCGGGGGAATCTGGGTGGGCATCCTGCTCCTCAACGGGNACCT
GCTCCTGAACACCTTCTCCGGCCCCTCCTGCCTCAGCTACAGGGTGGGCACGAARCCCTCGGCCTCGCT
CCGCTGGCACCAGGCACTGTACCTGCTGGARTTYTTCCTGCCACTGGCGCTCATCCTCTTTGCTATTGT
GAGCATTGGGCTCACCATCCGGAACCGTGGTCTGGGCGGGCAGGCAGGCCCGCAGAGGGCCATGCGTGT
NCTGGCCATGGTGGTGGCYGTCTACACCATCTGCTTCTTGCCCAGCATCATCTTTGGCATGGCTTCCAT
GGTGGCTTTCTGGCTGTCCGCCTGCCGATCCCTGGACCTCTGCACACAGCTCTTCCATGGCTCCCTGGC
CTTCACCTACCTCAACAGTGTCCTGGACCCCGTGCTCTACTGCTTCTCTAGCCCCAACTTCCTCCACCA
GAACCGGGCCTTGCTGGGCCTCACGCGGGGCCGGCAGGGCCCAGTGAGCGACGAAAGCTCCTACCAACC
CTCCAGGCAGTGGCGCTACCGGGAGGCCTCTAGGAAGGCGGARGCCATAGGGAAGCTGAAAGTGCAGGG
CGAGGTCTCTCTGGAAAAGGAAGGCTCCTCCCAAGGGCTGAAGGCCAGCTGCAGGGCTGCAGCGCTGTG
GGGGTAAGGGCTGCCGCGCTCTGGCCTGGARGGACAAGGCCAGCACACGGTGCCTCAACCAACTGGACA
AGGGATGGCGGCAGACCARGGGCCAGGCCAAAGCACTGGCAGGACTCAGGTGGGTGGCAGGKARARAAA
~- 13
GEI-70087 CA 02234414 1998-06-03
CCCACCTTAGGCCTCTCAGTGTGTCCAGGATGGCATTCCCAGAATGCAGGGGAGAGCAGGATGCCGGGT
GGAGGAGACAGGCAAGGTGCCGTTGGCACACCAGCTCAGACAGGGGCCTGCGCAGCTGCAGGGGACAGA
CGCCCAATCACTGTCACAGCAGAGTCACCTTAGAAATTGGACAGCTGCATGTTCTGTGCTCTCCAGTTT
GTCCCTTCCAATATTAATAAACTTCCCTTTTAAATATAUUVV;bb~A~AAAA
c A par~al m~ oticle s~ of a human HEOAD54 (SEQ ID NO: 3).
Table 4d
ARAAFLAPILALEFVLGLVGNSLALFIFCIHTRPWTSNTVFLVSLVAADFLLISNLPLRVDYYLLHETW
RFGAAACKVNLFMLSTNRKASVVFLTAIALNRYLKW XPHHVLNRASVGAXARVXGGIWVGILLLNGXL
LLNTFSGPSCLSYRVGTKPSASLRWHQALYLLEFFLPLALILFAIVSIGLTIRNRGLGGQAGPQRAMRV
LAMVVAVYTICFLPSIIFGMASMVAFWLSACRSLDLCTQLFHGSLAFTYLNSVLDPVLYCFSSPNFLHQ
NRALLGLTRGRQGPVSDESSYQPSRQWRYREASRKAEAIGKLKVQGEVSLEKEGSSQGLKASCRAAALW
G.GLPRSGLEGQGQHTVPQPTGQGMAADQGPGQSTGRTQVGGRXXNPP.ASQCVQDGIPRMQGRAGCRV
EETGKvpLAHQLRQGpAQLQGTDAQsLsQQsHLRNwTAAcsvLssLsLpILINFpFK~KKKKKK
5 d A partial amino acid se~l.,e~ of a human HEOAD54 (SEQ ID NO: 4).
The present ill~liol~ further relates to pol)~ 1r-~ that hybridize to the herein above-
~ In this regard, the present il~ )n especially relates to polyn~ ~ which
h~liJ~ under stringent conditions to the herein above~,~ d pol.~ ;.1~ As herein used, the
10 term "stringent calditions" means hyl~ l;a~ will occur ally if there is at least 80%, and pl~r~,~ly at
least 90~/O, and more plcrt;ldbly at least 95~/O, yet even more pl~,f~ bly 97-99% iderl~ity between the
s~l~J~
Pol~ c1~1;~ ofthe ill~ , which are identical or ~,--rr. ;- ..~lly identical to a ~
se~ r-~ ~ce ~ Y1 in SEQ ID NO: 1 or a r.~ ~..r.l1 thereof, may be used as hybri-1i7~ti-n probes for
l 5 cDNA and genanic DNA, to isolate full-lengdl cDNAs and genomic clones c ~ HEOAD54 and to
isolate cDNA and genanic clones of other genes (in -'- ' B genes ~ B ~ , and a i' ~' -~,s from
species other than human) that have a high s~~ sil,lil~ity to the HEOAD54 gene. Such
~,. ;-I; ~ 1 ;1 -- - ~ ' ~ 1!~ are known to those of sl~ll in the art. Typically these . .~ ,lw~ S~r ~ ~ are
80% i~l~nti~ ldbly 90% i-l~tic~1, more p~crc;l~ly 95% identical to that of the referent. The
probes generally will c~ e at least 15 n11l~1eQti-lf~. Preferably, such probes will have at least 30
nlll~l~ti~l~ and mayhave at least 50 .. 1~.1;.1f.s Pa~ ly plcirt;ll~ probes will range betvveen 3o
and 50 1...~ f ~;
. 14
',
GEI-70087 CA 02234414 1998-06-03
One embodiment, to obtain a pol~ encoding the HEOAD54 polypeptide, ' ' ,,
3s and Ol i' - ' ~~ from species other than human, C"" ~y~; ~ ' the steps of screening an al~pl~l idl~
library under stingent 1~1-- ;.li ,~1 ;- .. . conditi~s with a labeled probe having the SEQ ID NO: 1 or a
-h thereof(includingtila~ of SEQ ID NO: 3), and isolating full-length cDNA and genomic clones
5 ~ ,, said poly~ '~'lfV~ S~ e Such 1~- ;-li ~ A ;~ ~-- techniques are well known to those of skill
in the art. St~f nt 1.~ . ccnditions are as defined above or alt "~ ely conditions under
uvt;l-h~ht ;~ u~ at 42~C in a solution ~ 50% r I, 5xSSC (150mM NaCI, lSmM
tnsodium citrate), 50 mM sodium pl~ (pH7.6), 5x D~l~dl'~ solution, 10 % dextran sulfate, and
20 ~allJnll d~..dtu-~, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at
10 about65~C.
The pol~ l~ and pOl~Li~a ofthe present ill~ may be employed as research
leagents and materials for d;~ov~ly of ~, 1-.. - -h~ and ~ to animal and human disease.
Vectors, Host Cells, E~
The present il-~ also relates to vectors which c~ e a pol~ or polynu~l- ~ ;-1
ofthe pres~t iu~ , and host cells which are g~..,..~;r~ .IJ~lC~ with vectors ofthe illVW~ and
to the ~JIUdU~ of polypeptides of the ill~ i~ by recombinant i ' . Cell-free I . j- " 1 ~io.~
systems can also be e . '( ,_1 to produce such proteins using RNAs derived from the DNA CO~LIu ,1~ of
the present invention.
For l~ ~ ~ ' nt pro~l-c~--n, host cells can be g.~ y eng,llwlcd to i~ ul~te CeA~)lC~
systems or portions thereof for poly. .~ ~ of the present invention. ~udu~ of polymlc l~ ;~
into host cells can be effected by methods ~ il~cl in many standard l~ y m~ml~le, such as Davis
et aL, BASICMETHODSIN'MOLECUL~R BIOLOGY(1986) and Sa,lLl~1o~ et al.,MOLECUZAR
CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor T Ah~.. ,.~o. y Press, Cold Spring
25 Harbor, N.Y. (1989) suchascalciumphosphate1.,.. , r~ -,DEAE~an.. ~ rr~,1;cn,
trans~ection, microinjection, cationic lipid-mediated l,~ .r~ , el~l~ n, ~ h~,l;u~, scrape
loading, ballis~c introduc~on or in1~i~n
Re~,lc~e~i./e c r 1~ of appropriate hosts include bacterial cells, such as ~
.~i sl~L~I~I, E. coli, S~ ",,yces and Bacillus subhlis cells; fimgal cells, such as yeast cells and
30 Aspergillus cells; insect cells such as Drosophila S2 and S~odo~t~c,a Sf9 cells; animal cells such as
CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes .~ .u.. ~ cells; and plant cells.
GEI-70087 CA 02234414 1998-06-03
A great vanety of ~"y~ ;ol~ systems can be used. Such systems include, among others,
~L~ o~ , epi~m~l and virus-denved systems, e.g., vectors denved from bacterial F'~ ' from
ba~ ~ ~ ;o~ ., fiom t~ u)~ , from yeast t~ from inserhon Pl~~ , firom yeast ~ 71'.. :~
m~ frorn viruses such as baculoviruses, papova vin~es, such as SV40, vaccinia viruses,
5 ad~,. uvi-u~,es, fowl pox viruses, i~ viruses and retroviruse~s, and vectors derived from
combioa~ions thereof, such as those denved from plasmid and h~ ~ ~ genetic ~ such as
cosmids and phag~nids. The ~A~ ;7.7;1~ systems may contain con~ol regions that regulate as well as
~,,1"~1~ e,,*-~;~. Generally, any system or vector suitable to m~int~in' p..~ or express
~I.~--v~ s to produce a pol~ i~ in a host may be used. The a~)lu~
10 may be inserted into an ~ ,5;Ol~ systern by any of a variety of well-known and routine tP~ .1 ;- r ,~
such as, for ~ . 'e, those set ~rth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
For secrehon ofthe tIanslated protein into the lumen ofthe e-..1.~ ..IiC ~ ~.. into the
. '~ . space or into the I ~ ullnlcllt~ .u~ dc secrehon signals may be
15 ~41~Nl~ into the desired polylJcl~li(le. These signals maybe c~ to the poly~c~lidc orthey
may be L~.~ k,, signals.
If the HEOAD54 pol~l,c~ lc is to be ~ ,scd for use in screening assays, g~.lclally, it is
p~,f..-cd that the pol~Lide be produced at the surface of the cell. In this event, the cells may be
c.,~cd prior to use in the s.,lw~,ng assay. If the HEOAD54 polypeptide is secreted into the
20 m~1inm the medium can be r.,covc.cd in order to recover and purify the polypeptide; if produced
intrac~ rly, the cells must first be Iysed before the polypeptide is r~cuvc,cd.
HEOAD54 polypeptides can be ~~ovc cd and purified from " ~ ,t cell cultures by well-
known methods ~1 ' g n sulfate or etbanol precipitation, acid ~ .. anion or cahon
hy r ;u~ L~
25 affinityCll~ o~ .l.y,~dliu,~yla~ cl~ phyandlectin~ pl.y. Mostp~crcl~bly,
_igh ~, r."...~ liqjuid ~,L~ j-l.y- is e . '~i for ~ ;r.~ Well knowni t~ for
l~ ~ ld g prc~ins may be; . ~ dito l~....~ ~l~ ac,tive ~..r~ - whenthe poly~tilc is dc.~ cd
during isolation and or ~,... ;r.. ~;~,.,
30 n:~, Assays
This i ilVCl~ Ul~ also relates to the use of HEOAD54 polynllcl~l;~l~ for use as 11i~osh~
reagents. Detecti~ of a mutated form of the HEOAD54 gene ~ with a dy~; r... ,. ~ ;u~ ~ willi provide
16
' GH-70087 CA 02234414 1998-06-03
a f~ l ;c tool that can add to or define a ~ Of a disease or ~ -xp1 il .;1;1y to a disease which
results from ùnder~*,l~,on, over~*"c~ l or altered ~*/1~;.~ll of HEOAD54. Individuals carrying
""~ in the HEOAD54 gene may be detected at the DNA level by a variety of k~
Nucleic acids for ~1; ~ .~ may be obtained ~om a subject's cells, such as from blood, urine,
S saliva, ~issue biopsy or autopsy rnaleri~. The genomic DNA may be used direc~y ~r ~lf~n or may
be ~ ;r~ ~ylll.ai~lly by using PCR or other - ..l.l;r~ ' . prior to analysis. RNA or
cDNA may also be used in similar fashion. Deletions and ~Liol~ can be detected by a change in size
ofthe amplified product in ~ -q~ to the normal gen~pe. Point ~ c can be ~ ;rfxl by
L~ ;ng ~..l)l;r.~1DNAtolabeledHEOAD54..~ ~;Jes~ f ~ ~s Perfectlymatcheds~~
10 can be ~ 1 fiom ...~ 1,~ duplexes by RNase digestion or by .lil~ ~cs in melting
4 ~-~ ~. DNA ~r r~ "l~S may also be detected by alterations in el~LIo~ho~ c mobil~ty
of DNA fi ~ .... ~' ~ in gels, with or without ~ agents, or by direct DNA ~.~ .g See, e.g.,
Myers et al., Science (1985) 230: 1242. SF~l-Pnre changes at specific Ir~ticn~ may also be revealed by
nuclease protection assays, such as RNase and S11~1- hon or the I ' ~ cleavage method. See
C~tonetaL,ProcNadAcadSciUSA(1985)85:43971401.Inanother~ .. ho~l;-.. --~1 anarrayof
oligon~lr1p~ti~les probes colll~lisillg the HEOAD54 n--rleotide se~.~F .~e or L~"w,l~ thereof can be
constructed to conduct efficient S~ g of e.g., genetic m~t~tions Array terhnolngy methods are
well known and have general applicability and can be used to address a variety of q~lpsti~n~ in
~le ~ r genetics ;"~ g gene ~pl~ iOII, genetic linkage, and genetic variability. (See for
e~ M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
The .l: ~ ;c assays offer a process for ~ or determining a ~ 1;h;1ily to inf~F;~ion~
such as b~Ctp-r~ fungal, p,~ and vi~l i..r~,l;~ . p~uLiculally infiP~ n~ caused by HlV-l or HIV-
2; pain; cancers; anorexia; bulimia; astbma; Pd~ 's disease; acute healt failure; 1~
L~t~;~, urina~y l~ 4 Oal~l)ulùaL~, angina pectoris; Illyo~uJidl in~rction; ulcers; asthrna;
25 ~l1~g~F~; benign plua1~Lic h-y~;lLIu~ , and psy~l~ûLic and n~ isUl~;la, ;~ n~ anxiety,
a~ n~ ~ manic d~l~iaalull, deliriUTm~ , severe mental l~lald~LiUII and ly~ " such as
I T ~ .'s disease or Gilles dela Tourett's ayll~hull~ through ~lF~tFctir~n of mut~tion in the HEOAD54
gene by the methods ~.. il ~
In addition, ;. .r~ 4;--i-s such as b~CtFri~l fun~l, pl~a~all and viral inf~tir ns, particularly
30 ;. .r~ .-c caused by HIV-l or HlV-2; pain; cancers; dllu~,~, bulirnia; as~rna; P~uhil~ull's disease;
acute heart failure; Ly~t ~-~ -; hy~.~ n, urinaTy l~ ioll, Oal~u~al~ aTlgina pectoris;
yu~ ,1iùn; ulcers; asthrna; allergies; benign prostatic hy~lLIul)lly, and ~ay-,lwlic and
.. , , . . ~ . . , . . , , I, ~ . . .
GEI-70087 CA 02234414 1998-06-03
~Ic" ~ ,includinganxiety,~]~ a~manicd~.c,~ lP.li~illm, ~ 1;A severe
mel~ ,kU(~ iiVII and J~ s, such as IT~ 1.. 's disease or Gilles dela Tourett s ~y~llv - e, can
be ~ w ~ by methods ~ ..~ .ng ~ t~ P from a sample derived from a subject an
~b-~n- ,..~lly .l~l~cd or il~,l~sod level of the HEOAD54 polypeptide or HEOAD54 mRNA
S D~-eas~ or illcr~scd .,A~ ;Vll can be ~ll~ed at the RNA level using any of the mPthr~e well
known in the art for the .~ of poly.-~ e~ c~ such as~ for -py~mple~ pcR~ RT-pcR~
RNase p.ul~ 4 Northern blothng and other 1~ ;.1;,A1 ;on mPfh~le Assay techniques that can be
used to determine levels of a prvtein~ such as an HEOAD54, in a sample derived from a host are well-
knawn to those of skill in the art Such assay methods include radi~ s, ~ ;v~binding
10 assays, Western Blvt analysis and ELISA assays
Thus in another aspect, the present hl~clltivn relates to a li~po ~.,I;r kit for a disease or
sll~pecf~hility to a disease, particularly ;..r~ such as baf~PrjAl, filDgal, IJIVIV~I~ and viral
fi~ , pdllil..UIally infections caused by HlV-I or HIV-2; pain; cancers; anorexia; bulimia; asd~ma;
Pa hi~vll's disease; acute heart failure; Ly~t - -, Ly~l~l~iO~, urinary ICt~lltiUII, va~yuluaia,
15 angina pectoris; ~--yu~l~al iuE~u~.liv4 ulcers; asthma; allergies; benign prostatic LY~IL1U~JI1Y; and
p~-,L~Aic and ~ Gla~ including amciety, s~ .. ia, manic ~1GP1~ , flPlirillm
PntiA, severe men~ Liv~ and ~ly- L;", ~, such as Ih--fl;.~l.--.'a disease or Gilles dela Tvure~s
~ Ulll~, which Culll~
(a) an HEOAD54 pûly~ eu~ P, preferably the mlr,lPoti(lP S~IP~ e of SEQ ID NO 1, or a
La~l~ t thereof;
(b) a nllrleotiflP s~u~ ~r-e s ~ mPnt~ry to that of (a);
(c) an HEOAD54 poly~GI)ti~e, preferably the poly~Gl~tide of SEQ ID NO 2, or a fid~ .-L therevf;
or
(d) an antibody to an HEOAD54 polypeptide, preferably to the poly~,GI~tide of SEQ ID NO 2
25 It will be ay~lG~idLGd that in any such kit, (a), (b), (c) or (d) may cv ..p~ise a s~lhstAntiAl
u~
Chromosome Assays
The ~ ]~;~1~ SfJ~ -~ ofthe present hlvGlltion are also valuable for .,1~-, . sn, f
30 i.lf-~l;f.,~1;on Thes~~ eissrerifirAllytargetedtoandcanLylJlid~GwithapdlLi~ulallocationonan
individualhumandl.~ ,os~ e The _~ gofrel_vant~l~f ~to.l-l., . s-."~ iacco Ji~gtothe
present invention is an i~l~vlL~lt first step in cvll~,laLil~ those 5~ ,r-c with gene A~ d disease
1 8
CA 02234414 1998-06-03
G~I-70087
Once a ~~ has been mapped to a precise ~,L~ Iocation, the physical position ofthe
~JI,~f ~.~ ~ on the ~L1~ - can be cull-,hl#d with genetic m~p data. Such data are found, for
c . 'e, in V. M~l1Q;~ ., T.~l~f.. ;~ in Man (av7 'a~'- on line through Johns Hopkins
Ulf~ Welch Medical Library). The ~ )l~ip between genes and diseases that have been mapped
5 to the same ~,~,"~,,"~1 region are then id~ ;fi~i through linkage aTlalysis (~ nr~ of physically
adjacent genes).
The ~ l~s in the cDNA or genomic se~lu- -.ee between affected and ..I.-rr~ d individuals can
also be ~ A If a mllt~tinn is obs~. ~.~1 in some or all of the affected individuals but not in
any normal individuals, then the .,,..li.l;.~- is likely to be the causalive agent of the disea.,e.
~o~'
The ~I~Ai~s ofthe u~ L,oll or their r.~ or analogs thereof, or cells e*~l~ them
can also be used as ;.. -~ to produce ~ s ;~ r for the HEOAD54 pol~J~ide~.
15 The term "i..""-~~ c" means that the antibodies have ~- ~h~ 11 greater affir~ty for the
p~)ly~ti~s ofthe invention than their affinity for other related poly i~ in the prior art.
Antibodies generated against the HEOAD54 pol~lJlid~ can be obtained by a 1 ~n~ the
lides or epitope bearing r.~.. ,t~ analogs or cells to an animal, plcrcl~ly a ro.~ ., using
routine pl~Ls. For ~lc~ of n~ o-.~l antibodies, any t ' . ~ which provides alfLil~ '
pl~luu~ll by ~"~ u~c cell line cultures can be used. r n~ ' include the h~
(Kohler, G. and Milstei~ C., Nature ~1975) 256:495-497), the trioma h ' . r" the human B-cell
L~ ùll~ technique (Kozbor et al., Immunolo~y Todcy (1983) 4:72) and the EBV~ ull~a
technique (Cole et aL, MONOCLONAL ANTIBODES AND CANCER THERAPY, pp. 77-96, Alan
R Liss, Inc., 1985).
T,~ or the plUlU liUll of single chain ~nt-~ ~ ' (U.s. Patent No. 4,946,778) can also
be adapted to produce single chain antibodies to polypeptides ofthis ill~ LiUll. Also, t ~ - mice, or
other ul~lla ;..-,~ er m~mm~lc may be used to express l.~ ..I;l~J; .c
The above~ l ~ ~il c ' may be; . ~ ~J ~ to isolate or to identify clones ~ lcaalllg the
poly~i~e or to purify the polype~tides by affinity dll~ ".. ~t~ y.
3 0 ~ ' against HEOAD54 poly~ti~ may also be ernployed to treat; . . rr ~ ~c such as
b~n~l, fun~l, pl.,lu~ and viral ;~lfiF.,I;o~l~, particularly ;..rr~ l;""~ caused by HIV-l or HIV-2; pain;
cancers; anorexia; bulimia; as~na; Pau~illaull's discase; acute heart failure; Ly~t~ aiOll; hy~ ,lla;vll;
19
GH-70087 CA 02234414 1998-06-03
urinary l~ iUI~ Oa~lua~, angina pectoris; Illy~lial i,~.,ti~ ulcers; asthm~; allergies; benign
prosta~c Ly~ llu~hy; and pay~ ~c and ~ 6 I disorders, including anxiety, s~ pl ~.a, manic
d~l~a;.~ rillm, ~1. ---- .~1;~ severe mental rG~atiOI~ and d~ , such a~s Ih~ 's disease
or Gilles dela Tourett s ayl~ul~ among others.
s
Vaccines
Another a~spect ofthe invention relates to a method for in~lllrin~ an immllno~ iC~l
IGa~ù~e in a m~mm~l which ~ ;s~,s in.~ in~ the mqmm~l with the HEOAD54 poly~GI.Iide,
or a ~ t thereof, ~ to produce ~til,ody and/or T cell immune ~es~u,~se to protect said
10 animal from r ~jr~s such as b~rt~ri~l fun~, p,~ 0an and viIal ;..r~ nc p~u~ly ;..r~;u,~
caused by HIV-l or HlV-2; pain; cancers; anore~a; bulimia; as~na; P~ukillaull'a disease; acute heart
fi~ilure; h~ : ; hy~la;ul~, urinary l~,t~ tiùll, Oa~lua~, angina pectoris; lllyùc~
iù~, ulcens; as~ma; allergie~s; Wgn prostatic l1Y~GIllu~lly, and pay~,L~tic and neul~
~IdGla, ~1 ' B anxiety, S~ .GI~, manic J~l~a;~4 delirium, ~l~mrnti~, severe mental
15 ~G~Idtiùn and ~y~ c, such as Ih~ h~'s disease or Gilles dela Tourett's ay~llulll<,, among
others. Yet another aspect ofthe invention relates to a method of ;.. 1.. ;-.~ i.. ol~l lGa~onse
in a m~mm~l which culll~lises dGLvGlillg an HEOAD54 poly~ tide via a vector dilG~ lg
,;,a;ull of an HEOAD54 polynllclP!otirl~ in vivo in order to induce such an ~logir~l
rei,~o~e to produce antibody to protect said animal from diseases.
Further aspect of the invention relates to an immlm~l~r~l/vaCcine forrn~ tif-n
(~""po~ ir,n) which, when introduced into a m~mm~li n host, induces an immlmolo~ ical ,tial,ul,se
in that m~mm~l to an HEOAD54 polypeptide wherein the c~ )oaition co...l.. ;ceS an HEOAD54
polypeptide or HEOAD54 gene. The vaccine formlll~tif n may further cc Ill~,iae a suitable carrier.
Since the ~OAD54 polypeptide may be broken down in the st~m~rh, it is preferably ~ Gd
25 pal~lllGl~lly (inr~ ir~ s~b~t~i~f~c, ;~ s~ r, illtla~ US, illt.ad~ llal etc. injecti~n).
FormnlAti~nc suitable for pal~,llttilal a~ l ;on include aqueous and non-aqueous sterile
injection soh~tirlnc which may contain anti~Xi~ntc~ buffers, b~cteri~astAtc and solutes which render
the formlllAtirm instonic with the blood of the Iti~ k,llt, and aqueous and non-aqueous sterile
"~ which may include a~Cl~e ~ e agents or Ih;~Lf~ g agents. The formlll~ti--nc may be
30 yl~,sellttid in unit-dose or multi-dose CU~1A;~'- a, for f Y~mple~ sealed ampoules and vials and may be
stored in a freeze-dried cnn(~itif n ,~ui""g only the addition of the sterile liquid carrier i~"...e~l;A~rly
prior to use. The vaccine formlllAti( n may also include adjuvant systems for r...l-Al~.;..g the
. .
' Gll-70087 CA 02234414 1998-06-03
;... o ~;~ ;ly ofthe form~ tion, such as oil-in water syste ns and other systems known in the
art. The dosage will depend on the specific activity of the vaccine and can be readily ~~ ed by
routine e~c.;~
5 S~- ~ Ir, Assays
The HEOAD54 poly~li3e of the present ill~ iùll may be; r 1,( ~ ~i in a SCI~Gl~ process
for ~ q~ which bind the receptor and which activate (agonists) or inhibit activation of
t~) the ~ceptor pol~ Aiie ofthe present Ill~ iUII. Thus, poly~ i~s ofthe Ill~/t;llliUll may
also be used to assess the binding of small ~ s~ and ligands in, for; . ' ~, cells, cell-free
10 pl~ , chemical libraries, and nat-w~l product mix~res. These substrates and ligands may be
na~als~lb~candligandsormaybe :~u~ ual or r...~ ",....1 ;rs SeeColigan etal.,Current
Protocols in Immunolo~ 1(2):Chapter 5 (1991).
HEOAD54 poly~lfli~ are l~-r '~ I_ for many ' -'~ 1 r.. I;o~, including many
' -' g Accold gl~" it is desirous to find ~ - ~ q~U~ i and drugs which stimlll~t~ HEOAD54 on the
15 one hand and which can inhibit the function of ~OAD54 on the other hand. In general, agonists are
. k ,~ for ll.- -~q~ and l)luyLyla~lic l,w~ ~ for such conditions as ;~ ~fi~ n~ such as b~ctPri~l,
fu~ pl~ and viral r ~ , pa~ ul~lly ;--rr~ C caused by HIV-1 or HIV-2; pain; cancers;
anorexia; bulimia; as~na; PdlLi~l;s disease; acute healt failure; Lyy..t ...~ , h~y~ ;Oll, urina~y
retenticn; O~ yOl~L., angina pectoris; lllyuc~ud~ hv~, -wcers; asdlma; allergies; benign prostatic
20 Lyy~lLI~yLy;andy~y~ icand~w~ lol Id;solL~ E5anxiety~s~ ylll~ manic
d~yl~ ll, deliriurn, d~n~i~ severe mental l~u~Liul~ and dy~ ., such as Th..~ ,l...-'s disease
or Gilles dela Tourett~s syllw~ l~~ ~ : c~; may be ~ ~ for a variety of ll.~ l ;c and
yl~yLyl~Lic ywyo~ for such ' - as inf~tion.~ such as b~ l, fungal, plvttJd)all and viral
;"rr,,~ n~, ya~ ,uL~ly ;ll~ , caused by HIV-1 or H[V-2; pain; cancers; ~l ~c,,.-a, bulimia; asmma;
25 Pal~ disease;acuteheartfailure;Lyy~ ..,Lyy~ l,urina yl~ tiolr,o~ uyulu~is,
angina pectoris; lllyu~-LI illIiu~livl~ ulcers; asthma; ~llf~ c; benign prostatic LYY~ILIUY1IY, and
y~ydl~)~and~le~ '~ I ~i~l~l~, ' " y~anxiety, s~ vyl~ .li4manicd~yl~ ;u~ lf~liri~m,
df m~nti~, severe mental -~daLivl~ and ~y~ .f -: ~c, such as IT~ disease or Gilles dela Tourettls
~ general, such screening p-ucedw~ involve ylùlu ,~g ayylUIJlia~ cells which express the
receptor pGiyy~iyti~le ofthe present invention on the surface thereo~ Such cells include cells from
m~mm~lc, yeast, Drosophila or E. coli. Cells ~yl~ing the receptor (or cell ~ b~o~ e c~ g the
GEI-70087 CA 02234414 1998-06-03
~ ul) are tben u~ l with a test c~ to obscIve binding~ or ~ or
inhibition of a r.~ ;o--~l response.
One screel~ing i ' rlP includes the use of cells which express the receptor ofthis illV~l~iUI~
(for example, i. --~-f~ h ~1 CHO cells) in a system which ll~ pH or jr~ll~ r
5 calcium changes caused by receptor a~,liValiull. Inthis t~ " cu~ may be cu.~ l with
cells l,*Jl~.l~ the receptor p~ li~ ofthe present ill~liùl~. A second ...~ respcnse, e.g.,
signal i.;...~ , pH changes, or changes in calcium level, is then ll~ci~l to determine whether the
pateD~al C~ tl activates or inhibits the recc~tor.
Another method involves SCI~ ~ ,, for receptor ' ' r~ by ~' g ~ ~ itir~n or~10 ~l; . . ~l A ;u. ~ of receptor-mediabed cAMP and/or adenylate cyclase ~ m~ ti~ n Such a method in
volves
f ~ a eukaryotic cell with the receptor of this invention to express the receptor on the cell
surface. The cell is then cxposedto p~al ~ ; inthe presence ofthe reccptor ofthis ill~ iiUll.
The amoullt of cAMP ~ l ;o-- is then lll~7ulvd. If the poten~ 1 binds the receptor, and
thus inhibits receptor binding, the levels of receptor-.--c,l; ~ 1 cAMP, or adellyl~ cyclase, activity will~5 be reduced or ill~,l~.
Anvther method for ~f ~ agvnLsts or ~ 1 y; ...: .t~ for the receptor ofthe present invention is
the yeast based i ' ~' gy as tlc7~ d in U.S. Patent No. 5,482,835, illcul~uldted by ,t;r~lulce herein.
The assays may simply test binding of a c ~.,.1;.1_1~; co...l.v~ l wherein adll~ e to the
cells bearing the receptor is detected by means of a label directly or ill(lh~,~ly ~ o~ r,d with the
20 c~ lr, colll~,uu,ld or in an assay involving c~ n with a labeled ~,.~I.~;~or. Further, these
assays may test whether the c~ r, cvlll~ùund results in a signal g, .~ ed by activation of the
receptor, using ~ t~ systems a~ lU~ t~ to the cells bearing the receptor at their surfaces.
Tnhihitors of activation are generally assayed in the plt;.7cllce of a known agonist and the effect on
acti~aLiun by the agonist by the p~;7.,ll~,f; of the c~ cu. . ~pu~ 1 is observed.
Further, the assays may simply COIll~liS-, the steps of mixing a c~n~lit~te colll~uulld with a
solution c~ p. a HEOAD54 polypeptide to form a mixture, 111~-7~Uillg HEOAD54 activity in
the mixture, and co" .1-~ ~ ;. ~ the HEOAD54 activity of the mixture to a standard.
The HEOAD54 cDNA, protein and antibodies to the protein may also be used to cullLgule
assays for ~ .g the effect of added cvlllyvul~F7 on the pro~l--ction of HEOAD54 mRNA and
protein in cells. For ~ ~- rle, an ELISA may be constructed for ll~f ~ 1 ;llg secreted or cell
a~v. :~lrd levels of HEOAD54 protein using ""~noclon~l and polyclonal antibodies by ~7L~ldard
methods known in the art, and this can be used to discover agents which may inhibit or enhance the
- . . - . . ~ . . : . . . , . - ~
GH-70087 CA 02234414 1998-06-03
,
prah~ )n of HEOAD54 (also called ,..~t~ ",;~ or agonist, ~i"Je~,Li~,~,ly) from suitably
m~nir~ t~cellsortissues. Standardmethodsforc~ h~ p s~,l~,fingassaysarewell
od in the art.
F ,' of ~ HEOAD54~ t~ include~ ' or,insomecases,
5 n~ or pra~ins which are closely related to the ligand of the HEOAD54, e.g., a r. ~ . . ,l of
the ligand, or small ~ which bind to the receptor but do not elicit a response, so that the activity
ofthe receptor is prevented.
Thus in another aspect, the present invention relates to a scl.,~l g kit for id~ tiryi~lg
ag~ni~tC~ g~ t~ ligands"~t~ b~ eDzymes,etc.forHEOAD54polypepticles;or
10 com~ is which d~ ase or eDhance the pro~llrti~n of HEOAD54 polyl,clJt;~l~s, which
C~ ;'f,S:
(a) an HEOAD54 polyp~tide, preferably that of SEQrDNO:2;
(b) a l~c~,~.ll,;.. .~1 cell ~r~ ing an HEOAD54 poly~ Jtide, preferably that of SEQID NO:2;
(c) a celH--~ l~e e,~lcssing an HEOAD54pol~ )tide, pl~lably that of SEQID NO:2;or
15 (d) ~ltil ody to an HEOAD54 polypeptide, preferably that of SEQ ID NO: 2.
It will be ah,lc.,iaLcd that in any such kit, (a), (b), (c) or (d) may CO~ a SU~ ;A
C~ 1)~~ t
Prophylacbic and Therapeubic Methods
This iU~tiUII provides rnethods oftIeabng an ~1",o. ",~1, ' related to both an excess of
and i..~ r..: ~1 amounts of HEOAD54 activity.
If the activity of HEOAD54 is in excess, several ~pl~llcs are available. One ~l.l~h
'~"'l" ;.~ admin;~ to a subject an inhibitor cf~ (""1~o": ,;) as lh,.~ abovc .1P~.,. i1~d along
with ap1~ c~ 11y ~ep~~~le carrier in an amount effectiveto inhibit a.,Liv~ti.~ byblocking
25 binding of ligands to the HEOAD54, or by inhibit ng a second signal, and thereby alleviating the
ah.~ 1 condition.
~ another approach, soluble forms of HEOAD54 polypeptides still capable of binding the
ligand in co...l~c~ n with c ~log~ ouc HEOAD54 may be ~A~ Lc~cd. Typical eTnboflinlfnt~ of
such c~ c~ c~....l.. ;~e La~ of the HEOAD54 poly~c~tide.
30In still another approach, ~ s:~iOU of the gene f- .c~l;u~ c~ ge~ HEOAD54 can be
inhibited using c~ c~;~ion bloc~u-g terhniq~es. Known such te~hni~lues involve the use of
e sc~ f!~ either internally gcl~ld~ or s~alf~Lcly af~ c~cd. See, for exarnple,
23
.,
G~-70087 CA 02234414 1998-06-03
O'Connor, JNeurochem (1991) 56:560 in OliP~ y~ $ as .Antis~nc~ ;tu~ ~ of Gene
EAY1CSaIOIL CRC Press, Boca Raton, FL (1988). Altcllldti~cly, oli~..-~ eQti~l~s which form triple
helices with the gene can be supplied. See, for c~l,p'e, Lee ef al., Nucleic Acids Res (1979)
6:3073; Cooney ef al., Science (1988) 241 :456; Dervan et al., Science (1991) 251: 1360. These
5 l~ligom~r~ can be ?~h~ cd per se or the relevant OligOIlwla can be CA~ in vivo.
For trea~ng ~1,. ,~ " ", ,1 conditi~s related to an under~l~;o~ of HEOAD54 and its activity,
several ~I,IuaLl~s are a~so a~ Orw a~plu~~ . a~ninis~ to a subject a
Ih~ f~ y effective amou~ of a u~ pu~ which activates HEOAD54, i.e., an agonist as ~1~ ;1
above, in axnbination with a yl~ J~ lyaC__r~ - carrier, to thereby alleviate the, bn~
10 condition. A}tematively, gene therapy may be employedto &the ~ pr~lu~ ofHEOAD54 by the relevant cells in the subject. For example, a pol~"-~ ofthe i~ ll may be
1 for tAlJl~a;~ in a l.~ -- defective retroviral vector, as ,lisc-~ above. The retroviral
;~eo~hu~l-m--aythenbeisolatedandi~u~luc~lintoap~L~cel~ h~ witha
~troviral plasrnid vector c ~ - 1 RNA encoding a pol)~ lidc of the present ill~iUII such that ~w
p~,.L ~ cell now plUlh~Ce ~ ;..rr",;. ~--C viral particles containing the gene of interest. These producer
cells may be ~h . . . L --~l to a subject for ~ Y ~ cells in vivo and ~lc~;on of the polypcptide in
qvo. For O~ ,.. ûfgene therapy, see Chapter 20, Gene Therapy andofherMolecular Genefic-based
TherapeuticA~,oa~h~s, (and l~,f~,.~s citedtherein) inE~nan ~.c' ' ~n~ir~, T S1~rh~n and
A P Read, BIOS S-,;~iLc ~~ Ltd (1996).
Fonnulation and Admioistra~on
Peptides, such a's the soluble form of HEOAD54 pl~ly~llid~, and agonis~s and ~ "~
peptides or small -' l~, may be formu~ d in c ' ~ir~n with a suitable pl~A. ~ tir~l carrier.
Such r~ e a ~ lly effective amount ofthe pol~ 1e or cr~ and a
25 ~ ",~--1 ;-~lly a~ ' carrier or excipient. Such carriers include but are not limited to, saline,
buf~d saline, dex~ose, water, glycerol, ethanol, and r~ ...,1, .~1 ;. I~ thereof. Fc.. ,... ,1 A ;~ should suit the
mode of adm ~ion~ and is well wi~in the skill ofthe art. The ill~ tiUII filrther relates to
pl~ l packs and kits c~ , one or more c~ i filled with one or more ofthe
ingredielIts ofthe ~UI' ~' 4;- --~1 c~ ..,q,Oc;~ ofthe illVt;l~liUll.
Puly~i~li~ and other c~ q,u~ le ofthe present invention may be employed alone or in
with other c~ pù~ such as ll.- ~,~ ;c c~...q~
24
i
GEI-70087 CA 02234414 1998-06-03
~l~,f~ ~ forms of systemic a ~ of the ~ l cr... ~l~ A ;....~ include ;
typically by ~us injecti~. Other injecti~ routes, such as ~ "t ~ ,u~ ;.~.,.. ~. ~-l~r, or
;.~il.,~ . i~ .... -1, can be used. Alternative means for systemic admin~ ... include 1.~ 1 and
l""~ ". ~1 adm c~tinn using ~ ,-- h~ such as bile salts or fusidic acids or other ~ ~. In
5 add~on, if p~ly ~ 1 ~ in eni~ric or ~ -r ~ r~ ;n~ ~, Ola~ ti~ may also be~ possible. Adm ~ti- n ofthese c ~ may also be topical and/or l-r~li7f~ in the form of salves,
pastes, gels and the like.
The dosage range required depends ~ the choice of peptide, the route of ~- h . ~ n, the
nature ofthe r(.. l ~;.,.. the nature ofthe subject's ccndition, and the ju~ 1 ofthe attf- ~ g
10 p-~ ;- - . Suitable dosages, however, are in the range of O.l-lO0 llg/kg of subject. Wide ~ in
the needed dosage, however, are to be expected in view ofthe variety of cn~ available and the
differing e~ of various routes of admin ~A;"" For; , lo~ oral admini~tifm would be
e~torequirehigherdosagesthanadmin:~ by il~ luus' j~ 1ifn Variationsinthese
dosage levels can be adjustedusing standard; , I routines for ~ , as is well ~ r~uod in
15 theart.
Pùl~li~ used in IIG;~ can also be generat~,d e ..'lnp,f ..~o..~ly in the subject, in LIG~
".~ C often referredto as "gene therapy" as ~ ;l~ above. Thus, for ~ o" cells from a
subject may be ~.~ with a pol~ lr.~ such as a DNA or RNA, to encode a pol.~JG~ G eX
viw, and for example, by the use of a retroviral plasmid vector. The cells are then il~uced into the
20 subject.
F ,~ --. Cell E~1G~;U11
The receptors ofthe present ill~,.rliull are ~ "~ in either human embryonic kidney 293
(HEK293) cells or adherent dhfr CHO cells. To ... ~,~;-..;~ i receptor ~,*)l~a;Vll, typically all 5' and 3'
25 ~ regions (UTRs) are rernoved from the receptor cDNA prior to insertion into a pCDN or
pCDNA3 vector. The cells are ~ ~1 with individual receptor cDNAs by L~G~ and selected in
the presence of 400 mg/ml G41X. Afcer 3 weeks of s~o,lf~inn, ilJi~;dual clones are picked and ~ n~l
for further analysis. HEK293 or CHO cells ~ rr~,~ with the vector alone serve as negative controls.
To isolate cell lines stably ~*)lGS~Jlg the ill~ lual IG~ , about 24 clones are typically selected and
3 0 analyzed by Northern blot analysis. Receptor mRNAs are generally d~ect-Ll~ in about 50% of the
G418-resistant ck.~nes analyzed.
. . . , . . ~ , : .:
GEI-70087 CA 02234414 1998-06-03
Example 2 Ligand bar~c for binding and fim~n~l assays.
A bank of over 200 putative receptor ligands has been assembled for screening. The bank
~.. q";c.~ t~ansmi~s,h.. ~nfsandJ.~ v~;nfekncrwntoactviaahumanseven1.. ~ b~
(~I) receptor; ~lly o~ cu~q,u~ which may be putanve agonists for a human ~M
S ~r, non~n ~ 1;- ~, b ' l ~y active peptides for which a ...~.""".1;~ . L has not yet
been ;~-..";r.~l and ~----q u-~ not found in nature, but which activate ~M I~Jtul~ with urlknown
natuIal ligands. This bank is used to ini~ally screen the receptor for known ligands, using both
r,~ ;on~l (i.e . calcium, cAMP, mic~uyL.y~ - ....- ~ -, oocyte ~ uyL~ slc,,~/, etc, see below) as well ~
binding assays.
Example 3: Ligand Binding Assays
Ligand binding assays provide a direct metlLlod for a~ ~.g receptor ~' ~r. ~ ~Icgy and are
a~ Ll to a high ILuu~ format. The purified ligand for a receptor is " -I;o~ to high specific
activity (50-2000 Ci/mmol) for binding studies. A detem~ination is then made that the process of
15 ' - ' L -ling does not diminish the activity of the ligand towards its receptor. Assay ~ for
buffers, ions, pH and other modulators such as .~-~- lw~ f e are oy1;..~;~ to establish a w~ ' " - signal
to noise ratio for both ~--- ~~.b~ f and whole cell receptor sources. Forthese assays, specific receptor
binding is defined as total a ~ ~ ~ ~ ' radioactivity minus the radioactivity ~~l~ulc;d in the presence of an
excess of ""1~ ligand. Where possible, more tban one ~ Iigand is used to define
residual nf .,~ c binding.
F.---~'~ 4: F---.-~;o~1 Assay in Xenopus Oocytes
Capped RNA 1~ from linealized plasmidtemplates ~ ~;-.g the receptor cDNAs ofthe
i~ ,~iul~ are s~ ~ in vitro with RNA pol.~ , ~ in acc~,ldal~e with standard plU~hllC ~. In
25 vitro 1~ er . ;y1~; are ,~ - .J~l in water at a final cou. ~ 1 ;o~ ~ of 0.2 mg/ml. Ovarian lobes are
rcmoved from adult female toads, Stage V ~ lated oocytes are obt~in~l~ and RNA 1. ,....j~,. ;yt~i (10
~/o~ytc) are injected in a 50 nl bolus using a , ; ~ Two el~LI~)de voltage clamps
are used to measure the currents from individual Xenopus oocytes in response to agonist ~*JO:iUlC.
R~.dil~gs are made in Ca2+ free Balth's medium at room 1. ..l~ ...c. The Xenopus system can be
30 used to screen kno-wn ligands and li~suc/~ll extracts for a~liv~ g ligands.
. ~ . ,
GII-70087 CA 02234414 1998-06-03
,
Example 5: Miclu~ ;c Assays
Achvation of a wide vanety of ~o~J~y .~ systems results in e~u~l~)ll of srnall
amounts of acid from a cell. The acid formed is largely as a result ofthe .1l~,l~1---- I-~bolir~ activity
requiredto fuel the ;~d~cell~ r signaling process. The pH changes inthe media surrounding the cell are
5 very small but are r~ by the CYTOSENSOR u~ h - (~; 1~ l~r Devices Ltd., Menlo
Park, CA). The CYTOSENSOR is thus capable oftlf~tf~t~ the a~livdliul. of a receptor which is
coupled to an energy utilizing ;~d~ Sigl~ ~ pathway such as the G-prcte.in cûupled rec~ptor of
the present ill~w~
10 r 1 6: Extract/Cell S~ t --d S~l~ L
A large number of ,.., -"--- tl; .-, receptors exist for which there remains, as yet, no cognate
a~livd~il.g ligand (~gf ni~) Thus, active Ligands for these ~ )t~l~ may not be included within the
ligands banks as i.L .d;r~l to date. Accordingly, the 7TM receptor ofthe invention is also r.. l;.. ~lly
screened (using calcium, cAMP, microphy~ ~-- h~v~ oocyte cl~llu~ l ~ ,y, etc., fi~"- ~ l screens)
l 5 against tissue eAtracts to ider~fy na~al ligands. E tracts that produce positive r.... ,, ;. ~ e~ can
be 5~1" -d; ~lly ,~ f,~l;ol~tPd until an activahng ligand is isolated and i~L .~;
F . '- 7: Calcium and cAMP F~ l Assays
7TM 1~1.7 which a~e ~,Aylw~l in HEK 293 ceLls have been shown to be coupled
20 r.~ ytoa~,livdliullofPLCandcalciurn...~ d;~ and/orcAMP~ dl;....or~ '~' Basal
calcium levels in the HEK 293 cells in receptor-l . " ,yr~ ~1 or vector ~~trol cells were observed to be in
the normal, l00 nM to 200 nM, range. HEK 293 cells eA~ h~ 1~ ' lt l~clJtul~ are loaded with
fura 2 and in a single day more than 150 selected ligands or Lis,su~/cell extracts are t;vdh~dt~ for agonist
induced calcium mrl)ili~tir)n Similarly, HEK 293 cells CAyl~;~ g recombinant 1~l~ are evaluated
25 for the ~ Y~ or inhibition of cAMP ~ludu ,li~ using standard cAMP ~ d ;l ~ assays.
Agonists ~l~~liug a calcium 11~;~ or cAMP n-",,"~;O-~ are tested in vector con~ol cells to determine
if the response is unique to the l. d~ ~~ r ~ ~ ~I cells ./A~I~7~h~ receptor.
All pUblif'~l ;l~n~, inrl~lAinE but not limited to patents and patent applir~*rJn~, cited in this
30 sper,ifir~tir~n are herein ill~l~old~td by l~r~ltllce as if each individual publication were ~pecifir~lly
and individually in~lir~t~d to be illcol~ Id~d by .t;r~-ellce herein as thûugh fully set for~.
: . .
CA 02234414 1998-06-03
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT
(A) NAME: SMITHKLINE BEECHAM CORPORATION
(B) STREET: ONE FRANKLIN PLAZA
(C) CITY: PHILADELPHIA
(D) STATE OR PROVINCE: PA
(E) COUNTRY: USA
(F) POSTAL CODE: 19103
(ii) TITLE OF THE INVENTION: cDNA CLONE HEOAD54 THAT ENCODES
A HUMAN 7-TRANSMEMBRANE RECEPTOR
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: RATNER & PRESTIA
(B) STREET: P.O. BOX 980
(C) CITY: VALLEY FORGE
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19482
(v) COMPUTER-READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TO BE ASSIGNED
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/050,124
(B) FILING DATE: 18-JUN-1997
(viii) ATTORNEY/AGENT INFORMATION:
28
' GEI-70087 CA 022344l4 l998-06-03
- .
(A) NAME: PRESTIA, PAUL F
(B) REGISTRATION NUMBER: 23,031
(C) REFERENCE/DOCKET NUMBER: GH-70087
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-407-0700
(B) TELEFAX: 610-407-0701
(C) TELEX: 846169
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1594 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GACTATCCTC CCACTTCAGG GTTTCTCTGG GCTTCCATCT TGCCCCTGCT GAGCCCTGCT 60
TCCTCCTCTA CCAGCAGCAC AACCCCCAGG CTGGGCTCAG AGACCTCATG TGGTGGGATC 120
ACTCAGTACC CCGAGGCGGA GGGAAGGAGG GAGGGCTGCA GGGTTCCCCT TGGCCTGCAA 180
25 ACAGGAACAC AGGGTGTTTC TCAGTGGCTG CGAGAATGCT GATGAAAACC CCAGGATGTT 240
GTGTCACCGT GGTGGCCAGC TGATAGTGCC AATCATCCCA CTTTGCCCTG AGCACTCCTG 300
CAGGGGTAGA AGACTCCAGA ACCTTCTCTC AGGCCCATGG CCCAAGCAGC CCATGGAACT 360
TCATAACCTG AGCTCTCCAT CTCCCTCTCT CTCCTCCTCT GTTCTCCCTC CCTCCTTCTC 420
TCCCTCACCC TCCTCTGCTC CCTCTGCCTT TACCACTGTG GGGGGGTCCT CTGGAGGGCC 480
30 CTGCCACCCC ACCTCTTCCT CGCTGGTGTC TGCCTTCCTG GCACCAATCC TGGCCCTGGA 540
GTTTGTCCTG GGCCTGGTGG GGAACAGTTT GGCCCTCTTC ATCTTCTGCA TCCACACGCG 600
GCCCTGGACC TCCAACACGG TGTTCCTGGT CAGCCTGGTG GCCGCTGACT TCCTCCTGAT 660
CAGCAACCTG CCCCTCCGCG TGGACTACTA CCTCCTCCAT GAGACCTGGC GCTTTGGGGC 720
TGCTGCCTGC AAAGTCAACC TCTTCATGCT GTCCACCAAC CGCACGGCCA GCGTTGTCTT 780
35 CCTCACAGCC ATCGCACTCA ACCGCTACCT GAAGGTGGTG CAGCCCCACC ACGTGCTGAG 840
CCGTGCTTCC GTGGGGGCAG CTGCCCGGGT GGCCGGGGGA CTCTGGGTGG GCATCCTGCT 900
.CCTCAACGGG CACCTGCTCC TGAGCACCTT CTCCGGCCCC TCCTGCCTCA GCTACAGGGT 960
GGGCACGAAG CCCTCGGCCT CGCTCCGCTG GCACCAGGCA CTGTACCTGC TGGAGTTCTT 1020
CCTGCCACTG GCGCTCATCC TCTTTGCTAT TGTGAGCATT GGGCTCACCA TCCGGAACCG 1080
TGGTCTGGGC GGGCAGGCAG GCCCGCAGAG GGCCATGCGT GTGCTGGCCA TGGTGGTGGC 1140
CGTCTACACC ATCTGCTTCT TGCCCAGCAT CATCTTTGGC ATGGCTTCCA TGGTGGCTTT 1200
CTGGCTGTCC GCCTGCCGCT CCCTGGACCT CTGCACACAG CTCTTCCATG GCTCCCTGGC 1260
29
' GEI-70087 CA 022344l4 l998-06-03
CTTCACCTAC CTCAACAGTG TCCTGGACCC CGTGCTCTAC TGCTTCTCTA GCCCCAACTT 1320
CCTCCACCAG AGCCGGGCCT TGCTGGGCCT CACGCGGGGC CGGCAGGGCC CAGTGAGCGA 1380
CGAGAGCTCC TACCAACCCT CCAGGCAGTG GCGCTACCGG GAGGCCTCTA GGAAGGCGGA 1440
GGCCATAGGG AAGCTGAAAG TGCAGGGCGA GGTCTCTCTG GAAAAGGAAG GCTCCTCCCA 1500
GGGCTGAGGG CCAGCTGCAG GGCTGCAGCG CTGTGGGGGT AAGGGCTGCC GCGCTCTGGC 1560
CTGGAGGGAC AAGGCCAGCA CACGGTGCCT CAAC 1594
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 amino acids
(B) TYPE: amino acid
(C)~ STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Leu Cys His Arg Gly Gly Gln Leu Ile Val Pro Ile Ile Pro Leu
1 5 10 15
Cys Pro Glu His Ser Cys Arg Gly Arg Arg Leu Gln Asn Leu Leu Ser
20 25 30
Gly Pro Trp Pro Lys Gln Pro Met Glu Leu His Asn Leu Ser Ser Pro
35 40 45
Ser Pro Ser Leu Ser Ser Ser Val Leu Pro Pro Ser Phe Ser Pro Ser
50 55 60
Pro Ser Ser Ala Pro Ser Ala Phe Thr Thr Val Gly Gly Ser Ser Gly
65 70 75 80
Gly Pro Cys His Pro Thr Ser Ser Ser Leu Val Ser Ala Phe Leu Ala
j 30 85 90 95
Pro Ile Leu Ala Leu Glu Phe Val Leu Gly Leu Val Gly Asn Ser Leu
1 100 105 110
Ala Leu Phe Ile Phe Cys Ile His Thr Arg Pro Trp Thr Ser Asn Thr
115 120 125
35 Val Phe Leu Val Ser Leu Val Ala Ala Asp Phe Leu Leu Ile Ser Asn
130 135 140
Leu Pro Leu Arg Val Asp Tyr Tyr Leu Leu His Glu Thr Trp Arg Phe
145 150 155 160
Gly Ala Ala Ala Cys Lys Val Asn Leu Phe Met Leu Ser Thr Asn Arg
165 170 175
Thr Ala Ser Val Val Phe Leu Thr Ala Ile Ala Leu Asn Arg Tyr Leu
180 185 190
' GH-70087 CA 02234414 1998-06-03
- ' .
Lys Val Val Gln Pro His His Val Leu Ser Arg Ala Ser Val Gly Ala
195 200 205
Ala Ala Arg Val Ala Gly Gly Leu Trp Val Gly Ile Leu Leu Leu Asn
210 215 220
5 Gly His Leu Leu Leu Ser Thr Phe Ser Gly Pro Ser Cys Leu Ser Tyr
225 230 235 240
Arg Val Gly Thr Lys Pro Ser Ala Ser Leu Arg Trp His Gln Ala Leu
245 250 255
Tyr Leu Leu Glu Phe Phe Leu Pro Leu Ala Leu Ile Leu Phe Ala Ile
0 260 265 270
Val Ser Ile Gly Leu Thr Ile Arg Asn Arg Gly Leu Gly Gly Gln Ala
275 280 285
Gly Pro Gln Arg Ala Met Arg Val Leu Ala Met Val Val Ala Val Tyr
290 295 300
15 Thr Ile Cys Phe Leu Pro Ser Ile Ile Phe Gly Met Ala Ser Met Val
305 310 315 320
Ala Phe Trp Leu Ser Ala Cys Arg Ser Leu Asp Leu Cys Thr Gln Leu
325 330 335
Phe His Gly Ser Leu Ala Phe Thr Tyr Leu Asn Ser Val Leu Asp Pro
340 345 350
Val Leu Tyr Cys Phe Ser Ser Pro Asn Phe Leu His Gln Ser Arg Ala
355 360 365
Leu Leu Gly Leu Thr Arg Gly Arg Gln Gly Pro Val Ser Asp Glu Ser
370 375 380
25 Ser Tyr Gln Pro Ser Arg Gln Trp Arg Tyr Arg Glu Ala Ser Arg Lys
385 390 395 400
Ala Glu Ala Ile Gly Lys Leu Lys Val Gln Gly Glu Val Ser Leu Glu
405 410 415
Lys Glu Gly Ser Ser Gln Gly
420
!
(2) INFORMATION FOR SEQ ID NO: 3:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1435 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
, .
' GEI-70087 CA 022344l4 l998-06-03
.
GGCACGAGCT GCCTTCCTGG CACCAATCCT GGCCCTGGAG TTTGTCCTGG GCCTGGTGGG 60
GAACAGTTTG GCCCTCTTCA TCTTCTGCAT CCACACGCGG CCCTGGACCT CCAACACGGT 120
GTTCCTGGTC AGCCTGGTGG CCGCTGACTT CCTCCTGATC AGCAACCTGC CCCTCCGCGT 180
GGACTACTAC CTCCTCCATG AGACCTGGCG CTTTGGGGCT GCTGCCTGCA AAGTCAACCT 240
CTTCATGCTG TCNACCAACC GCAAGGCCAG CGTTGTCTTC CTCACAGCCA TCGCACTCAA 300
CCGCTACCTG AAGGTGGTGC ANCCCCACCA CGTGCTGAAC CGTGCTTCCG TGGGGGCANC 360
TGCCCGGGTG GNCGGGGGAA TCTGGGTGGG CATCCTGCTC CTCAACGGGN ACCTGCTCCT 420
GAACACCTTC TCCGGCCCCT CCTGCCTCAG CTACAGGGTG GGCACGAARC CCTCGGCCTC 480
GCTCCGCTGG CACCAGGCAC TGTACCTGCT GGARTTYTTC CTGCCACTGG CGCTCATCCT 540
CTTTGCTATT GTGAGCATTG GGCTCACCAT CCGGAACCGT GGTCTGGGCG GGCAGGCAGG 600
CCCGCAGAGG GCCATGCGTG TNCTGGCCAT GGTGGTGGCY GTCTACACCA TCTGCTTCTT 660
GCCCAGCATC ATCTTTGGCA TGGCTTCCAT GGTGGCTTTC TGGCTGTCCG CCTGCCGATC 720
CCTGGACCTC TGCACACAGC TCTTCCATGG CTCCCTGGCC TTCACCTACC TCAACAGTGT 780
CCTGGACCCC GTGCTCTACT GCTTCTCTAG CCCCAACTTC CTCCACCAGA ACCGGGCCTT 840
GCTGGGCCTC ACGCGGGGCC GGCAGGGCCC AGTGAGCGAC GAAAGCTCCT ACCAACCCTC 900
CAGGCAGTGG CGCTACCGGG AGGCCTCTAG GAAGGCGGAR GCCATAGGGA AGCTGAAAGT 960
GCAGGGCGAG GTCTCTCTGG AAAAGGAAGG CTCCTCCCAA GGGCTGAAGG CCAGCTGCAG 1020
GGCTGCAGCG CTGTGGGGGT AAGGGCTGCC GCGCTCTGGC CTGGARGGAC AAGGCCAGCA 1080
CACGGTGCCT CAACCAACTG GACAAGGGAT GGCGGCAGAC CARGGGCCAG GCCAAAGCAC 1140
20 TGGCAGGACT CAGGTGGGTG GCAGGKARAR AAACCCACCT TAGGCCTCTC AGTGTGTCCA 1200
GGATGGCATT CCCAGAATGC AGGGGAGAGC AGGATGCCGG GTGGAGGAGA CAGGCAAGGT 1260
GCCGTTGGCA CACCAGCTCA GACAGGGGCC TGCGCAGCTG CAGGGGACAG ACGCCCAATC 1320
ACTGTCACAG CAGAGTCACC TTAGAAATTG GACAGCTGCA TGTTCTGTGC TCTCCAGTTT 1380
GTCCCTTCCA ATATTAATAA ACTTCCCTTT TAAATATAAA A~iUUUU~AAA AAAAA 1435
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 476 amino acids
( B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ala Arg Ala Ala Phe Leu Ala Pro Ile Leu Ala Leu Glu Phe Val Leu
1 5 10 15
Gly Leu Val Gly Asn Ser Leu Ala Leu Phe Ile Phe Cys Ile His Thr
20 25 30
Arg Pro Trp Thr Ser Asn Thr Val Phe Leu Val Ser Leu Val Ala Ala
35 40 45
!
' GEI-70087 CA 02234414 1998-06-03
' ' '
Asp Phe Leu Leu Ile Ser Asn Leu Pro Leu Arg Val Asp Tyr Tyr Leu
50 55 60
Leu His Glu Thr Trp Arg Phe Gly Ala Ala Ala Cys Lys Val Asn Leu
65 70 75 80
Phe Met Leu Ser Thr Asn Arg Lys Ala Ser Val Val Phe Leu Thr Ala
85 90 95
Ile Ala Leu Asn Arg Tyr Leu Lys Val Val Xaa Pro His His Val Leu
100 105 110
Asn Arg Ala Ser Val Gly Ala Xaa Ala Arg Val Xaa Gly Gly Ile Trp
0 115 120 125
Val Gly Ile Leu Leu Leu Asn Gly Xaa Leu Leu Leu Asn Thr Phe Ser
130 135 140
Gly Pro Ser Cys Leu Ser Tyr Arg Val Gly Thr Lys Pro Ser Ala Ser
145 150 155 160
lS Leu Arg Trp His Gln Ala Leu Tyr Leu Leu Glu Phe Phe Leu Pro Leu
165 170 175
Ala Leu Ile Leu Phe Ala Ile Val Ser Ile Gly Leu Thr Ile Arg Asn
180 185 190
Arg Gly Leu Gly Gly Gln Ala Gly Pro Gln Arg Ala Met Arg Val Leu
195 200 205
Ala Met Val Val Ala Val Tyr Thr Ile Cys Phe Leu Pro Ser Ile Ile
210 215 220
Phe Gly Met Ala Ser Met Val Ala Phe Trp Leu Ser Ala Cys Arg Ser
225 230 235 240
Leu Asp Leu Cys Thr Gln Leu Phe His Gly Ser Leu Ala Phe Thr Tyr
245 250 255
Leu Asn Ser Val Leu Asp Pro Val Leu Tyr Cys Phe Ser Ser Pro Asn
260 265 270
Phe Leu His Gln Asn Arg Ala Leu Leu Gly Leu Thr Arg Gly Arg Gln
275 280 285
Gly Pro Val Ser Asp Glu Ser Ser Tyr Gln Pro Ser Arg Gln Trp Arg
290 295 300
Tyr Arg Glu Ala Ser Arg Lys Ala Glu Ala Ile Gly Lys Leu Lys Val
305 310 315 320
Gln Gly Glu Val Ser Leu Glu Lys Glu Gly Ser Ser Gln Gly Leu Lys
325 330 335
Ala Ser Cys Arg Ala Ala Ala Leu Trp Gly Gly Leu Pro Arg Ser Gly
340 345 350
Leu Glu Gly Gln Gly Gln His Thr Val Pro Gln Pro Thr Gly Gln Gly
355 360 365
Met Ala Ala Asp Gln Gly Pro Gly Gln Ser Thr Gly Arg Thr Gln Val
370 375 380
33
.. . , , , . . j . :
. GEI-70087 CA 02234414 1998-06-03
Gly Gly Arg Xaa Xaa Asn Pro Pro Ala Ser Gln Cys Val Gln Asp Gly
385 390 395 400
Ile Pro Arg Met Gln Gly Arg Ala Gly Cys Arg Val Glu Glu Thr Gly
405 410 415
Lys Val Pro Leu Ala His Gln Leu Arg Gln Gly Pro Ala Gln Leu Gln
420 425 430
Gly Thr Asp Ala Gln Ser Leu Ser Gln Gln Ser His Leu Arg Asn Trp
435 440 445
Thr Ala Ala Cys Ser Val Leu Ser Ser Leu Ser Leu Pro Ile Leu Ile
0 450 455 460
Asn Phe Pro Phe Lys Tyr Lys Lys Lys Lys Lys Lys
465 470 475
34
