Highly conserved aspartate 68, tryptophane 73 and glycine 109 in the N-terminal extracellular domain of the human VIP receptor are essential for its ability to bind VIP.
The human VIP receptor belongs to a subfamily of G protein-coupled receptors that includes secretin, glucagon and several other receptors. We have used site-directed mutagenesis to investigate the requirement of highly conserved aspartate 68 (mutant D68G), tryptophane 73 (mutant W73G), proline 87 (mutant P87G), glycine 109 (mutant delta 109) and tryptophane 110 (mutant W110G) for the ability of the human VIP receptor to bind VIP. After transfection of mutated cDNAs in Cos-7 cells, it appeared that mutants G87P and W110G bound VIP with the same dissociation constant as the wild type receptor whereas mutants W73G, P87G and delta 109 did not bind VIP. Since all mutated receptor proteins were synthesized by Cos-7 cells (Western blot) and expressed at the plasma membrane level (immunofluorescence studies), it is concluded that the N-terminal extracellular domain of the human VIP receptor contains highly conserved amino acid residues which are essential for its intrinsic binding activity.
Couvineau
A
A
Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, INSERM U 410, Faculté de Médecine Xavier Bichat, Paris, France.
Gaudin
P
P
Maoret
J J
JJ
Rouyer-Fessard
C
C
Nicole
P
P
Laburthe
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D005455:Fluorescent Antibody Technique; D005998:Glycine; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018005:Receptors, Vasoactive Intestinal Peptide / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014364:Tryptophan; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D018005:Receptors, Vasoactive Intestinal Peptide; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014660:Vasoactive Intestinal Peptide; D014364:Tryptophan; D005998:Glycine