Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure.
Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (<Pro>294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the <Pro>294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.
Vertongen
P
P
Department of Biochemistry and Nutrition, School of Medicine, Université Libre de Bruxelles, Bât G/E, CP 611, 808 route de Lennik, B-1070, Bruxelles, Belgium.
Solano
R M
RM
Juarranz
M G
MG
Perret
J
J
Waelbroeck
M
M
Robberecht
P
P
D000262:Adenylyl Cyclases / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002478:Cells, Cultured; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology
D000262:Adenylyl Cyclases; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D008024:Ligands; D011392:Proline; D018005:Receptors, Vasoactive Intestinal Peptide; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014157:Transcription Factors; D014660:Vasoactive Intestinal Peptide; C513109:TCF15 protein, human