Presence of a N-terminal signal peptide in class II G protein-coupled receptors: crucial role for expression of the human VPAC1 receptor.
The hVPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) has an N-terminal signal peptide like all other class II G protein-coupled receptors (GPCRs). We determined the role of the signal peptide in expression of human VPAC1 receptor in transfected CHO cells. Three constructs were transfected: Flag30-hVPAC1, a receptor containing an inserted FLAG sequence between Ala30 and Ala31 and fused in the C-terminal position to GFP; Flag30-[delta1-30]-hVPAC1, the same construct as Flag30-hVPAC1 but lacking the 1-30 putative signal peptide (SP) sequence; Flag0-hVPAC1, a receptor containing an N-terminal FLAG sequence and fused in the C-terminal position to GFP. For each construct, we determined 125I-VIP binding, VIP-induced cAMP production, GFP fluorescence and indirect immunofluorescence on nonpermeabilized cells incubated with mouse monoclonal anti-Flag antibodies. The data were consistent with a crucial role of the signal peptide for expression of functional VPAC1 receptors at the cell surface and suggested that the signal peptide is cleaved during the translocation of the receptor to the plasma membrane, probably in the endoplasmic reticulum.
Couvineau
Alain
A
INSERM U410, Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, Paris F-75018, France. coucou@bichat.inserm.fr
Rouyer-Fessard
Christiane
C
Laburthe
Marc
M
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D021382:Protein Sorting Signals; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP