Different vasoactive intestinal polypeptide receptor domains are involved in the selective recognition of two VPAC(2)-selective ligands.
A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-terminal histidine by hexanoic acid (C(6)-VIP), behaved as a VPAC(2) preferring agonist in binding and functional studies on human VIP receptors, and radioiodinated C(6)-VIP was a suitable ligand for binding studies on wild-type and chimeric receptors. We evaluated the properties of C(6)-VIP, its analog AcHis(1)-VIP, and the VPAC(2)-selective agonist Ro 25-1553 on the wild-type VPAC(1) and VPAC(2) receptors and on the chimeric receptors exchanging the different domains between both receptors. VIP had a normal affinity and efficacy on the chimeras starting with the amino-terminal VPAC(2) receptor sequence. The binding and functional profile of these chimeric receptors suggested that the high affinity of Ro 25-1553 for VPAC(2) receptors is supported by the amino-terminal extracellular domain, whereas the ability to prefer C(6)-VIP over VIP is supported by the VPAC(2) fifth transmembrane (TM5)-EC(3) receptor domain. These results further support the hypothesis that the central and carboxyl-terminal regions of the peptide (modified in RO 25-1553) recognize the extracellular amino-terminal region domain, whereas the amino-terminal VIP amino acids bind to the TM receptor core. VIP had a reduced affinity and efficacy on the N-VPAC(1)/VPAC(2) and on the N-->EC(2)-VPAC(1)/VPAC(2) chimeric receptors. C(6)-VIP behaved as a high-affinity agonist on these constructions. The antagonists [AcHis(1),D-Phe(2),Lys(15),Arg(16), Leu(27)]VIP(3-7)/GRF(8-27) and VIP(5-27) had comparable affinities for the wild-type receptors and for the two latter chimeras, supporting the hypothesis that these chimeras were properly folded but unable to reach the high-agonist-affinity, active receptor conformation in response to VIP binding.
Juarranz
M G
MG
Department of Biochemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium.
Van Rampelbergh
J
J
Gourlet
P
P
De Neef
P
P
Cnudde
J
J
Robberecht
P
P
Waelbroeck
M
M
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000494:pharmacology; D011487:Protein Conformation; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*
D008024:Ligands; D010456:Peptides, Cyclic; D018005:Receptors, Vasoactive Intestinal Peptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011994:Recombinant Proteins; C089777:Ro 25-1553; D014660:Vasoactive Intestinal Peptide