A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector.
The stimulatory effect of VIP on intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimeric VPAC(1)/VPAC(2), or mutated receptors. The VIP-induced [Ca(2+)](i) increase was linearly correlated with receptor density and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar VPAC(2) receptor density. The study was performed to establish the receptor sequence responsible for that difference. VPAC(1)/VPAC(2) chimeric receptors were first used for a broad positioning: those having the third intracellular loop (IC(3)) of the VPAC(1) or of the VPAC(2) receptor behaved, in that respect, phenotypically like VPAC(1) and VPAC(2) receptor, respectively. Replacement in the VPAC(2) receptor of the sequence 315-318 (VGGN) within the IC(3) by its VPAC(1) receptor counterpart 328-331 (IRKS) and the introduction of VGGN in state of IRKS in VPAC(1) was sufficient to mimic the VPAC(1) and VPAC(2) receptor characteristics, respectively. Thus, a small sequence in the IC(3) of the VPAC(1) receptor, probably through interaction with G(alphai) and G(alphaq) proteins, is responsible for the efficient agonist-stimulated [Ca(2+)](i) increase.
Langer
Ingrid
I
Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, B-1070 Brussels, Belgium. ilanger@ulb.ac.be
Vertongen
Pascale
P
Perret
Jason
J
Waelbroeck
Magali
M
Robberecht
Patrick
P
D000262:Adenylyl Cyclases / Q000378:metabolism; D000331:Aequorin / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism* / Q000494:pharmacology*
D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D014660:Vasoactive Intestinal Peptide; D000331:Aequorin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D002118:Calcium