Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor

Abstract

The VPAC 2 receptor, as all members of the G-protein-coupled receptor (GPCR)-B family, has two highly conserved motifs in the third intracellular (IC 3 ) loop: a lysine and a leucine located at the amino-terminus and two basic residues separated by a leucine and an alanine at the carboxyl-terminus. This study evaluates the involvement of those conserved amino acid sequences in VPAC 2 signal transduction and regulation. The residues were mutated into alanine and mutants were expressed in Chinese hamster ovary (CHO) cells stably transfected with G 16 and aequorin. Mutation of L310 reduced efficacy of vasoactive intestinal polypeptide (VIP) to stimulate adenylate cyclase activity through G s coupling by 75%, without affecting VIP capability to stimulate an increase in [Ca 2+ ] i through G 16 coupling. Mutation of R325 and, to a lesser extend, K328 reduced VIP efficacy to stimulate [Ca 2+ ] i increase and VIP potency to stimulate adenylate cyclase. The combination of mutations of both amino-and carboxyl-terminus located conserved motifs of the IC 3 loop generates an inactive receptor with respect to [Ca2+] i increase and adenylate cyclase activation, but also with respect to receptor phosphorylation and internalization that were indeed directly correlated with the potency of inactivation of the receptors.The amino-terminus of the VPAC 2 receptor IC 3 loop is thus involved in adenylate cyclase activation and the carboxyl-terminus of the IC 3 loop participates in both G s and G 16 coupling. The mutations studied also reduced both receptor phosphorylation and internalization in a manner that appeared directly linked to the alteration of G s and G 16 coupling.

Mechanisms involved in VPAC receptors activation and regulation: lessons from pharmacological and mutagenesis studies

“…In VPAC 2 receptors, L 310 located in the proximal part of IC3 contributes to Gαs activation and the proximal part of IC3 (R 325 and K 328 ) is involved in both Gαs and Gα16 coupling. The combined mutations of these three amino acids generates an inactive VPAC 2 receptor with respect to [Ca 2 + ] i increase and adenylate cyclase activation (Langer et al, 2005). …”

“…Absence of inhibitory effect of PKA and PKC inhibitors (Langer et al, 2005; Langlet et al, 2005), use of dominant negative GRK and GRK over-expression experiments (Shetzline et al, 2002; Huang et al, 2007), suggest that GRK are the main kinases involved in VIP receptors phosphorylation. VPAC 1 receptor phosphorylation induces β-arrestin translocation to cell membrane, however, over-expression of β-arrestin in HEK 293 cells only causes a minor decrease in cAMP, suggesting VPAC 1 desensitization may occur through an arrestin-independent mechanism (Shetzline et al, 2002).…”

Differential determinants for coupling of distinct G proteins with the class B secretin receptor

“…Within the third intracellular loop, mutation of a central lysine-leucine motif of the opossum parathyroid hormone receptor, rat GLP1 receptor, and human VPAC1 and VPAC2 receptors, analogous to the human secretin receptor residues Lys 302 -Leu 303 (ICL3A), results in defects in agonist-stimulated cAMP production, calcium mobilization, and ligand binding to varying degrees (1,7,25,38,45,62). In the present work, replacing these residues with alanines in the human secretin receptor resulted in normal ligand binding and calcium signaling, but reduced cAMP responses.…”

MOLECULAR EVOLUTION OF GPCRS: Secretin/secretin receptors

“…In ICL2, Arg241, and Lys242 have been reported to be critical for the inositol phosphate (IP 3 ) signaling in many secretin GPCR members (Mathi et al 1997, Langer et al 2005. However, mutation analysis showed that this motif did not affect the calcium responses in human SCTR (Garcia et al 2012).…”