title: Identification of cytoplasmic domains of hVPAC1 receptor required for activation of adenylyl cyclase. Crucial role of two charged amino acids strictly conserved in class II G protein-coupled receptors.
abstract: The VPAC1 receptor mediates the action of two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide. It is a class II G protein-coupled receptor-activating adenylyl cyclase (AC). The role of the N-terminal extracellular domain of hVPAC1 receptor for VIP binding is now established (Laburthe, M., Couvineau, A. and Marie, J. C. (2002) Recept. Channels 8, 137-153), but nothing is known regarding the cytoplasmic domains responsible for AC activation. Here, we constructed a large series of mutants by substituting amino acids with alanine in the intracellular loops (IL) 1, 2, and 3 and proximal C-terminal tail of the receptor. The mutation of 40 amino acids followed by expression of mutants in chinese hamster ovary cells showed the following. (i) Mutations IL1 result in the absence of expression of mutants, suggesting a role of this loop in receptor folding. (ii) All residues of IL2 can be mutated without alteration of receptor expression and AC response to VIP. (iii) Mutation of residues IL3 points to the specific role of lysine 322 in the efficacy of the stimulation of AC activity by VIP. This efficacy is reduced by 50% in the K322A mutant. (iv) The proximal C-terminal tail is equipped with another important amino acid since mutation of glutamic acid 394 reduces AC response by 50%. The double mutant K322A/E394A exhibits a drastic reduction of >85% in the efficacy of VIP in stimulating AC activity in membranes and cAMP response in intact cells without alteration of receptor expression or affinity for VIP. These data highlight the role of charged residues in IL3 and the proximal C-terminal tail of hVPAC1 receptor for agonist-induced AC activation. Because these charged residues are absolutely conserved in class II receptors for peptides, which are all mediating AC activation, they may play a general role in coupling of class II receptors with the Gs protein.
authors.affiliation: INSERM U410 Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, F-75018 Paris, France. coucou@bichat.inserm.fr
authors.forename: Alain
authors.initials: A
authors.lastname: Couvineau
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Lacapere
authors.forename: Yossan-Var
authors.initials: YV
authors.lastname: Tan
authors.forename: Christiane
authors.initials: C
authors.lastname: Rouyer-Fessard
authors.forename: Pascal
authors.initials: P
authors.lastname: Nicole
authors.forename: Marc
authors.initials: M
authors.lastname: Laburthe
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D018005:Receptors, Vasoactive Intestinal Peptide / Q000235:genetics / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D000262:Adenylyl Cyclases