Contribution of the carboxyl terminus of the VPAC1 receptor to agonist-induced receptor phosphorylation, internalization, and recycling.
When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC1 receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser250 (in the second intracellular loop), Thr429, Ser435, Ser448 or Ser449, and Ser455 (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.
Langlet
Christelle
C
Laboratoire de Chimie Biologique et de la Nutrition, Faculté de Médecine, Université Libre de Bruxelles, Bruxelles B-1070, Belgium.
Langer
Ingrid
I
Vertongen
Pascale
P
Gaspard
Nathalie
N
Vanderwinden
Jean-Marie
JM
Robberecht
Patrick
P
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000737:chemistry; D011992:Endosomes / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D008985:Monensin / Q000737:chemistry; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014660:Vasoactive Intestinal Peptide / Q000737:chemistry
D000911:Antibodies, Monoclonal; D008024:Ligands; D010455:Peptides; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide; D012694:Serine; D008985:Monensin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases