300

Molecular Cloning, mRNA Distribution and Pharmacological Characterization of a VIP/PACAP Receptor in the Frog

Rana ridibunda

D. ALEXANDRE,

a

Y. ANOUAR,

a

S. JÉGOU,

a

A. FOURNIER,

b

AND H. VAUDRY

a,c

a

European Institute for Peptide Research (IFRMP 23), Lab. Cell. Mol. Neuroendocrinol., INSERM U413, University of Rouen, 76821 Mont-Saint-Aignan, France

b

INRS-Institut Armand Frappier, Université du Québec, Pointe Claire, Canada H9R1G6

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activatingpolypeptide (PACAP) are two closely related neuropeptides that belong to the secre-tin/glucagon/growth hormone-releasing hormone family of peptide hormones.

1,2

The effects of PACAP and VIP are mediated through three distinct G protein-coupled receptor subtypes: the VPAC

1

and VPAC

2

receptor types (VPAC

1

-R andVPAC

2

-R) that bind VIP and PACAP with equal affinity,

3

and the PAC

1

receptortype that selectively binds PACAP with high affinity.

4

These receptors show bothoverlapping and complementary distributions in the mammalian central nervous sys-tem and peripheral tissues.

1–4

Cloning VIP/PACAP receptors from distant species should prove useful in estab-lishing the structure–activity relationships of these receptors and elucidating theevolutionary process of this family of receptors. In this respect, the frog modelappears very appropriate, given the pivotal position of amphibians in the phylogenyof vertebrates.

Screening of a frog pituitary cDNA library with degenerate primers deduced frommammalian VIP/PACAP receptor sequences made it possible to isolate a cDNAclone encoding a 444-amino acid preprotein, that we termed fPVR,

5

which exhibitsthe greatest homology with human VPAC

1

-R (63

%

identity) but possesses also sig-nificant similarity with VPAC

2

-R (53

%

identity).To determine the pharmacological profile of the fPVR, the cDNAs encoding the

frog receptor or the human VPAC

1

-R were expressed in the LLC-PK1 cell line. Asexpected, both PACAP and VIP were potent stimulators of cAMP production byfPVR- and human VPAC

1

-R-transfected cells (see

F

IGURE

1). However, secretincould not stimulate cAMP production in fPVR-transfected cells (

F

IG

.

1A) althoughthis peptide is known to selectively stimulate VPAC

1

-R (

F

IG

.

1B) but not VPAC

2

-Ractivity.

3

Thus, on the basis of its pharmacological characteristics, the fPVR appearsto be more closely related to the human VPAC

2

-R than to the VPAC

1

-R.Reverse transcriptase-PCR analysis revealed that fPVR mRNA is more widely

expressed in peripheral tissues than VPAC

1

-R or VPAC

2

-R (see

F

IGURE

2A).

In situ

c

Telecommunication: Voice: (33) 235 14 66 24; fax: (33) 235 14 69 46.hubert.vaudry@univ-rouen.fr

301ALEXANDRE

et al.

: VIP/PACAP RECEPTOR IN THE FROG

FIGURE 1. Effect of PACAP-related peptides on cAMP concentration in LLC-PK1cells transiently expressing the fPVR (A) or the human VPAC1-R (B). Transfected cellswere incubated with graded concentrations of mammalian PACAP38 (�), porcine VIP (�)and porcine secretin (�). Data are expressed as the mean ± SEM of three (PACAP and VIP)or two (secretin) independent experiments performed in triplicate.

302 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 2. Distribution of fPVR mRNA. A. Analysis of fPVR expression in varioustissues of the frog Rana ridibunda by RT-PCR. RNA from the tissues indicated was incu-bated in the presence (RT+) or absence (RT–) of reverse transcriptase. The cDNAs obtainedwere then amplified by PCR using specific fPVR primers to generate DNA products of 369bp. The lane labeled fPVR corresponds to a PCR performed on the fPVR cDNA clone usedas a positive control. Autoradiographs of Southern blots performed on the PCR productsand hybridized with the fPVR cDNA are shown. B. In situ hybridization analysis of fPVRmRNA in the frog brain and pituitary gland using a specific cRNA probe. The autoradio-graphs show the distribution of fPVR mRNA in frog brain hemisections at the level of thetelencephalon and diencephalon. AD, anterodorsal segmental nucleus; BON, basic opticnucleus; CO, subcommissural organ; DP, dorsal pallium; Dst, dorsal striatum; LP, lateralpallium; LS, lateral septum; MP, medial pallium; MS, medial septum; NA, nucleus accum-bens; NDB, nucleus of the diagonal band of Broca; NMLF, nucleus of the medial longitudi-nal fasciculus; NPM, nucleus profundus mesencephali; NPv, nucleus of the periventricularorgan; PtG, pretectal gray; PtrG, pretoral gray; VH, ventral hypothalamic nucleus; Vst, ven-tral striatum; III, oculomotor and trochlear nuclei; 6, tectal lamina six.

303ALEXANDRE

et al.

: VIP/PACAP RECEPTOR IN THE FROG

hybridization histochemistry on frog brain showed that fPVR mRNA is highlyexpressed in several thalamic and hypothalamic nuclei including the central thalamicnucleus, the ventromedial thalamus, the anterior preoptic area, the ventral hypothal-amus, and the nucleus of the periventricular organ. The fPVR gene is also expressedin the pallium and striatum albeit at lower levels (

F

IG

.

2B). Such a distribution isreminiscent of those of VPAC

1

-R and VPAC

2

-R of mammals.To conclude, we have isolated a frog VIP/PACAP receptor that exhibits the high-

est sequence homology with the mammalian VPAC

1

-R but possesses pharmacolog-ical and tissue distribution characteristics of both VPAC

1

-R and VPAC

2

-R ofmammals. The cloning of this frog receptor provides the molecular basis for theidentification of the structural determinants for ligand binding and activation ofPACAP/VIP receptors.

ACKNOWLEDGMENTS

This research was supported by INSERM (U 413), an INSERM-FRSQ exchangeprogram to A.F. and H.V., and the Conseil Régional de Haute-Normandie. H.V. isAffiliated Professor at the INRS-Institute Armand Frappier (Pointe Claire, Canada).

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