Table Of ContentPACAP and
VIP Receptors
Edward J. Goetzl*, Julia K. Voice and Glenn Dorsam
ImmunologyandAllergy,UniversityofCalifornia,SanFrancisco,RoomUB8B,Box0711,533Parnassus
Avenue, San Francisco, CA 94143-0711, USA
*corresponding author tel: 415-476-5339, fax: 415-476-6915, e-mail: [email protected]
DOI: 10.1006/rwcy.2000.23009.
SUMMARY BACKGROUND
This subfamily of G protein-coupled receptors Discovery
consists of PAC , which binds PACAP alone, and
1
VPAC1 and VPAC2, which bind PACAP and VIP Vasoactive intestinal peptide (VIP) and pituitary
with equal affinity. These homologous receptors all adenylate cyclase-activating peptide (PACAP) are
have a long N-terminal extracellular sequence with constituents of one structural superfamily of neu-
five conserved cysteines, as well as conserved other roendocrine hormones, which also includes secretin,
cysteinesandbasicaminoacidsinthefirstandsecond glucagon, glucagon-like peptide, and growth hor-
extracellular loops and third intracellular loop and mone-releasing hormone or factor. VIP and PACAP
cytoplasmic tail. There are numerous splice variants are linked by some functional similarities and by
of PAC1, but not of the VPAC receptors. As for the their sharing of two of the three receptors in one
corresponding ligands, these receptors are distributed subfamily of G protein-coupled cellular receptors
preferentially in the nervous system, endocrine and (GPCRs) (Table 1) (Harmar et al., 1998). The three
immuneorgans,intestinesandlungs.Signalingthrough VIP/PACAP receptors were cloned initially by PCR-
two or more G proteins is mediated principally by based and GPCR hybridization techniques.
increases in [cAMP] and [Ca2(cid:135)]. Genetic manipula-
i i
tionofexpressionofthereceptorsis nowinprogress.
Table 1 Structures and encoding genes of the PACAP and VIP receptors
Receptor subtype Gene (HUGO) Chromosomal location Size (aa)
(IUPHAR nomenclature)
Human Other
PAC ADCYAP1R1 7p14 4 (rat) 495 (rat)
1
VPAC VIPR1 3p22 8 (rat) 457 (human)
1
9 (mouse) 437 (mouse)
VPAC VIPR2 7q36.3 4 (rat) 437 (human)
2
12 (F2 mouse)
2250 Edward J. Goetzl et al.
Alternative names Chromosome location and linkages
PAC was formerly designated the type I PACAP The gene encoding rat PAC has been isolated and
1 1
receptor.VPAC hadbeentermedthetypeIIPACAP characterized, demonstrating a single copy and a
1
receptor and the type I VIP receptor. VPAC had complex structure similar to those of genes for other
2
beentermedthetypeIIIPACAPreceptorandtypeII membersofthissubfamilyofGPCRs.RatPAC gene
1
VIP receptor. spans 40kb and contains 15 exons (Chatterjee et al.,
1997). Introns are 320 bp to 10.5 kbp and exhibit
Structure splicephasingoftypes0,1,and2.Allspliceacceptor–
donor sequences conform to the GT/AG consensus
rule.Thesizesandorganizationofexonsandintrons,
PAC ,VPAC ,andVPAC GPCRsshowahighlevel
1 1 2 and the intron–exon boundaries resemble those of
of homology to each other and share structural
genes encoding GPCRs for secretin, glucagon, and
features, such as a long N-terminus and short loops.
parathyroid hormone. Unlike VPAC and VPAC ,
1 2
forwhichnosplicevariantshavebeenidentified,PAC
Main activities and 1
showsextensivealternativesplicinginseveraldomains
pathophysiological roles (Pisegna and Wank, 1996; Pantaloni et al., 1996;
Chatterjee et al., 1996; 1997). Two alternative exons
PAC has been cloned from rat, mouse, bovine, and intheregionencodingthethirdintracellularloophave
1
humancells(PisegnaandWank,1993;Harmaret al., beentermedhipandhop,andthelatterexistsashop1
1998). PAC of each species binds PACAP-27 and and hop2 due to different splice acceptor sites. The
1
PACAP-38 (IC =1nM) with 1000-fold higher affi- hip-hop variants differ in signaling properties.
50
nity than VIP, and exhibits even lower affinities for Alternative splicing of exons equivalent to hip and
PHI(peptidehistidineisoleucine),PHV(peptidehisti- hop1 in human PAC1 leads to four variants desig-
nated null, SV-1, SV-2, and SV-3, that also differ in
dine valine), secretin, and growth hormone-releasing
transductionalabilities.Fourothervariantsarisefrom
factor (GRF) (Ohtaki et al., 1998).
VPAC hasbeenclonedfromrat,human,andmouse differential use of alternative exons in the 50 untrans-
1
cells (Ishihara et al., 1992; Harmar et al., 1998). lated region of the rat PAC1 gene. A splice variation
VPAC ofeachspeciesbindsPACAP-27,PACAP-38, that leads to a 21 amino acid deletion in the extra-
1
and VIP with equal affinity (IC =0.2–1nM), but cellularN-terminusandanotherwithsequencediffer-
50
differences exist among species in the rank-order of encesintransmembranedomainsIIandIVappearto
affinity of binding of other closely related peptides. represent one type of mechanism responsible for dif-
VPAC has been cloned from rat, mouse, and ferent binding affinities and signaling potencies of
2
human cells (Lutz et al., 1993; Inagaki et al. 1994; PACAP-38andPACAP-27.Thereisonecopyeachof
Harmar et al., 1998). VPAC2 of each species binds the VPAC1 and VPAC2 genes, and no alternatively
PACAP-38 and VIP with equal affinity (IC =2– spliced forms have been identified for either subtype.
50
4nM), but has slightly lower affinity for PACAP-27. Human VPAC1 spans 22kbp and is composed of 13
exons of 42bp to 1400bp and 12 introns of 0.3–
6.1kbp(Sreedharanetal.,1995).Thestructureofthe
GENE
gene encoding VPAC has not been delineated com-
2
pletely, but chromosomal localization is known
Accession numbers
(Mackay et al., 1996).
Althoughitseemsreasonabletoassumethatexpres-
See Table 2. sion of each receptor is regulated transcriptionally
Table 2 Accession numbers for PAC , VPAC , and VPAC
1 1 2
PAC VAPC VPAC
1 1 2
Human D17516 U11079, U11080, U11081, U11082, U11083,
U11084, U11085, U11086, U11087
Rat D14908, D14909 AF059678 U09631, Z25885
Mouse S82970 S82966
PACAP and VIP Receptors 2251
and with tissue specificity, few definitive studies have secretin receptor. Dissociation of ligand-binding
addressed these questions. One notable contribution affinity from ligand potency in transduction of
is the demonstration that corticosteroids suppress responses for some chimeras emphasized the likely
levels of mRNA encoding VPAC in cultured lung complexityofcompositionofcompletereceptorunits.
1
cells and reduce signals reported by VPAC 50-flank- Another series of chimeras of nonidentical compo-
1
ing sequence luciferase constructs introduced by nents of human and rat VPAC , and selected site-
1
transfection (Pei, 1996). The 50-glucocorticoid nega- directed mutants of both, revealed that the difference
tive response element was that mapped to a 126bp in rat VPAC high-affinity binding and human
1
sequence containing a glucocorticoid receptor-bind- VPAC low-affinity binding of PHI is attributable
1
ing site between (cid:255)36 and (cid:255)21bp from the transcrip- tothreeaminoacidsinthefirstextracellularloopand
tion start site. Promoters of each gene are now being adjacent third transmembrane domain (Couvineau et
defined fully with respect to other functional al., 1996).
elements.
Cell types and tissues expressing
PROTEIN
the receptor
Description of protein
The distinctive patterns of tissue distribution of each
PACAP/VIP receptor to date have been mapped
PAC , VPAC , and VPAC are related seven
1 1 2 principally by radioligand binding and semiquantifi-
transmembranedomainGPCRs.Withineachspecies,
cation or probe detection of encoding mRNA (Table
amino acid sequence identity among the three
3). The attendant problems of any one approach and
receptors is 49% to 51%, and the similarity of each
the possibility of dissociations between the amounts
with the GPCRs for secretin, glucagon,glucagon-like
of mRNA and protein limit confidence in the initial
peptide I, and growth hormone-releasing hormone
results. Nonetheless, it is clear that VPAC and
ranges from 34% to 47%. As for most subfamilies of 1
VPAC are often expressed with reciprocal densities
GPCRs,thegreatestdegreeofhomologyiswithinthe 2
in one type of cell, tissue representation may be
transmembrane domains and least in the N- and C-
complementaryasintheratCNS(Usdinetal.,1994),
terminal segments. Receptors of this subfamily share
and both show high levels of inducibility and
other structural features, which as yet have not been
repressibility. One such example is in a cell line
related to receptor function, including long N-
model for thymocytes, where nearly exclusive expres-
terminal extracellular sequences with five conserved
sion of VPAC changes to much higher expression of
cysteines, two additional conserved cysteines located 1
VPAC and lower levels of VPAC within hours of
one each in the first and second extracellular loops, 2 1
exposure to antigen and antigen-presenting cells
and conserved basic amino acids in the third
(Pankhaniya et al., 1998). Antibodies specific for
intracellular loop and cytoplasmic tail, that may
each receptor in the subfamily now have become
facilitate coupling to G .
s available, which should permit definitive analyses of
tissue-specific expression and the determinants of
Affinity for ligand(s)
regulation of such expression.
A few aspects of the structural determinants of
SIGNAL TRANSDUCTION
specific ligand binding have been elucidated for the
VPAC receptor. A series of chimeras of the rat
1
VPAC and secretin receptors were constructed PAC ,VPAC ,andPVAC eachcoupletomultipleG
1 1 1 2
involving exchanges of the N-terminus, first extra- proteins, resulting in concurrent initiation of diverse
cellular loop or both (Holtmann et al., 1995). The signaling pathways (Table 3). G is presumed to
s
native receptors bound their respective ligands with mediate stimulation of adenylate cyclase and an
2nMK valuesandcrossboundtheother ligandwith increase in [cAMP], that is the hallmark of cellular
d i
K values three orders of magnitude higher (Table 3). effects of VIP and PACAP. In addition to evidence
d
WhentheVPAC N-terminuswassubstitutedforthat for a physical association between G and VPAC ,
1 s 1
of the secretin receptor, ligand binding and transduc- specific suppression of G by transfection of G (cid:11)
s(cid:11) s
tion of biological responses typical of VPAC were chain antisense plasmids with a hygromycin-resis-
1
observed. For reciprocal conversion of VPAC to a tance element into VPAC -expressing epithelial cells,
1 1
secretinreceptor, however,the chimerarequiredboth followed by hygromycin selection, blunted VIP-
the N-terminus and the first extracellular loop of the induced increases in [cAMP] (Goetzl et al., 1993).
i
2252 Edward J. Goetzl et al.
Table 3 Binding, signaling, tissue distribution, and pharmacology of PAC1, VPAC1, and VPAC
2
Receptor K (nM) Responses Predominant tissue Selective
d
(EC , nM) of expression
50
[cAMP] [Ca2(cid:135)] Agonists Antagonists
i i
PAC 1 0.1–1.6 10–50 CNS (olfactory bulb, thalamus, Maxadilan PACAP6-38
1
hypothalamus, hippocampus,
cerebellum)
Adrenal medulla
Macrophages
VPAC 0.2–1 0.3–1 2.5 CNS (cerebral cortex, VIP1,7/GRF VIP3,7/GRF
1
hippocampus) VIP4-28
Lung
Gastrointestinal tract
Liver
Prostate
Macrophages and lymphocytes
VPAC 3–10 10–30 27 CNS (thalamus, suprachiasmatic Ro25-1553 VIP4-28
2
nucleus, hippocampus, brainstem, Ro25-1392
spinal cord, dorsal root ganglia)
Skeletal and cardiac muscle
Gastrointestinal tract
Kidney
Adipose tissue
Testes
Macrophages and lymphocytes
VIP1,7/GRF,[K15R16L27]VIP(1-7)GRF(8-25)-NH ;VIP3,7/GRF,[Ac-His1D-Phe2Lys15Arg16]VIP(3-7)GRF(8-27)-NH ;Ro25-1553,
2 2
Ac-His1-[Glu8Lys12Nle17Ala19Asp25Leu26Lys27;28Gly29;30Thr31]-NH VIP(cyclo21-25);andRo25-1392,
2
Ac-His1[Glu8OCH -Tyr10Lys12Nle17Ala19Asp25Leu26Lys27;28]VIP(cyclo21-25).
3
That similar antisense suppression of G led to activation of L-type Ca2(cid:135) channels (Chatterjee et al.,
i2(cid:11)
marked enhancement of VIP-induced increases in 1996).
[cAMP],suggestedthatG hasanegativeregulatory
i i2
effectonVIPenhancementofadenylatecyclase.Each
receptor also activates phosphoinositide-specific
DOWNSTREAM GENE
phospholipase C (PLC), with resultant elevation of
[Ca2(cid:135)]. The greater potency of PACAP-38 than ACTIVATION
i
PACAP-27 in PAC -mediated activation of PLC and
1
elevationof[Ca2(cid:135)] hasbeenattributedtoa21amino There are several reports of VIP or PACAP
i
acid segment of the N-terminal extracellular domain, enhancing or suppressing proliferation or functional
basedonresultsofstudiesofaseriesofsplicevariants responses of normal or neoplastic cells in vitro
(Pantaloni et al., 1996). The G protein is assumed (Ogasawara et al., 1997; Maruno et al., 1998). It is
q/11
to couple the receptors to PLC, based on data from assumedthatpartofthecapacityofVIPandPACAP
other systems, but this has not been demonstrated to influence growth is attributable to alterations in
directly. One transmembrane domain IV splice transcription of immediate early genes critical for
variant of PAC failed to activate either adenylate proliferative responses. However, no studies to date
1
cyclase or PLC, but stimulated Ca2(cid:135) influx by have focused on this important question.
PACAP and VIP Receptors 2253
BIOLOGICAL CONSEQUENCES in insulin-secreting cells. Proc. Natl Acad. Sci. USA 91,
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