Mutational analysis of cysteine residues within the extracellular domains of the human vasoactive intestinal peptide (VIP) 1 receptor identifies seven mutants that are defective in VIP binding.
We have used site-directed mutagenesis to investigate the requirement of cysteine residues in the extracellular domains of the human VIP 1 receptor for binding VIP. Cys37, Cys50, Cys63, Cys72, Cys86, Cys105 and Cys122 (N-terminal extracellular domain), Cys208 and Cys215 (second extracellular loop) and Cys288 (third extracellular loop) were mutated into glycine, and mutated cDNAs transfected into Cos-7 cells. It appeared that mutants C50G, C63G, C72G, C86G, C105G, C122G, and C288G did not bind VIP whereas mutants C37G, C208G and C215G bound VIP with the same dissociation constant (#0.5 nM) as the wild-type receptor. All mutated receptor proteins were synthesized by Cos-7 cells (Western blot) and delivered at the plasma membrane level (confocal microscopy). The fact that C208G and C215G mutants retained a complete binding activity while the C288G mutant was inactive does not suggest the presence of a functionally relevant disulfide bond between the second and third extracellular loop of the human VIP receptor contrary to what has been shown in several other heptahelical receptors. It is also concluded that the six crucial cysteine residues, e.g., Cys50, Cys63, Cys72, Cys86, Cys105 and Cys122 in the N-terminal extracellular domain, may be functionally important by forming intramolecular disulfide bonds which help to maintain the topology for ligand binding in human VIP 1 receptors.
Gaudin
P
P
Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, INSERM U 410, Faculté de Médecine Xavier Bichat, Paris, France.
Couvineau
A
A
Maoret
J J
JJ
Rouyer-Fessard
C
C
Laburthe
M
M
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics*; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D007834:Lasers; D008566:Membranes / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018005:Receptors, Vasoactive Intestinal Peptide / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D018005:Receptors, Vasoactive Intestinal Peptide; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide; D003545:Cysteine