Asn229 in the third helix of VPAC1 receptor is essential for receptor activation but not for receptor phosphorylation and internalization: comparison with Asn216 in VPAC2 receptor.

After stimulation with agonist, G protein coupled receptors (GPCR) undergo conformational changes that allow activation of G proteins to transduce the signal, followed by phosphorylation by kinases and arrestin binding to promote receptor internalization. Actual paradigm, based on a study of GPCR-A/rhodopsin family, suggests that a network of interactions between conserved residues located in transmembrane (TM) domains (mainly TM3, TM6 and TM7) is involved in the molecular switch leading to GPCR activation. We evaluated in CHO cells expressing the VPAC(1) receptor the role of the third transmembrane helix in agonist signalling by point mutation into Ala of the residues highly conserved in the secretin-family of receptors: Y(224), N(229), F(230), W(232), E(236), G(237), Y(239), L(240). N(229)A VPAC(1) mutant was characterized by a decrease in both potency and efficacy of VIP stimulated adenylate cyclase activity, by the absence of agonist stimulated [Ca(2+)](i) increase, by a preserved receptor recognition of agonists and antagonist and by a preserved sensitivity to GTP suggesting the importance of that residue for efficient G protein activation. N(229)D mutant was not expressed at the membrane, and the N(229)Q with a conserved mutation was less affected than the A mutant. Agonist stimulated phosphorylation and internalization of N(229)A and N(229)Q VPAC(1) were unaffected. However, the re-expression of internalized mutant receptors, but not that of the wild type receptor, was rapidly reversed after VIP washing. Receptor phosphorylation, internalization and re-expression may be thus dissociated from G protein activation and linked to another active conformation that may influence its trafficking. Mutation of that conserved amino acid in VPAC(2) could be investigated only by a conservative mutation (N(216)Q) and led to a receptor with a low VIP stimulation of adenylate cyclase, receptor phosphorylation and internalization. This indicated the importance of the conserved N residue in the TM3 of that family of receptors.

Mutation of the phosphorylatable residue Thr429 in Glu of the human VPAC1 led to a constitutively desensitized receptor.

The hVPAC1 receptor is rapidly phosphorylated and internalized by agonists but not re-expressed at the membrane after washing. Mutation of Ser/Thr residues in the C-terminus reduced phosphorylation but not internalization that was abolished only when all the phosphorylatable residues were mutated. Substitution of Thr429 by Glu mimicking a phosphothreonin led to a mutant with unchanged binding properties, decreased coupling to adenylate cyclase consisting in a reduced VIP potency, increased basal and VIP stimulated phosphorylation, preserved internalization followed by a rapid receptor re-expression. These are the expected characteristics of a constitutively desensitized receptor, putting forward the role of Thr429 phosphorylation in that process.

Comparative efficacy of VIP and analogs on activation and internalization of the recombinant VPAC2 receptor expressed in CHO cells.

Using a monoclonal antibody interacting with the extracellular amino-terminus of the human VPAC2 receptor but that did not interfere with ligand binding, we measured by flow cytometry receptor internalization and trafficking induced by full agonists, partial agonists and an antagonist in Chinese hamster ovary cells expressing the recombinant receptor. The agonists, but not the antagonist, induced a rapid, dose-dependent receptor internalization blocked by hypertonic sucrose that was more pronounced for the VIP analog N-hexanoyl-VIP (80%) than for VIP and Ro 25-1553 (50%) and the [A11]-VIP (20%). Re-expression of the receptors at the membrane was achieved within two hours after exposure to VIP and Ro 25-1553 was blocked by 25 microM monensin but not by 10 microg/ml cycloheximide. Re-expression was much slower after exposure to the acylated peptide and was blocked by preincubation with 25 microM monensin and 10 microg/ml cycloheximide.

Hexanoylation of a VPAC2 receptor-preferring ligand markedly increased its selectivity and potency.

We synthesized a VIP analog that combines mutations that decrease the affinity for the VPAC1 receptor but maintain a high affinity for the VPAC2 receptor with an amino-terminal hexanoylation that increases the affinity for the VPAC2 receptor with a limited decrease in the affinity of the VPAC1 receptor. The resulting Hexanoyl[A19,K(27,28)]VIP had the expected properties of a high affinity for the VPAC2 receptor and a low affinity for the VPAC1 receptor and also a low affinity for the PAC1 and secretin receptors. With a 1000-fold preference for the VPAC2 receptor and a IC50 value of binding of 1 nM, this compound is the most potent and the most selective agonist presently described.

Evidence for a direct interaction between the Thr11 residue of vasoactive intestinal polypeptide and Tyr184 located in the first extracellular loop of the VPAC2 receptor.

We developed previously VPAC(1) [vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptor]>VPAC(2) receptor selective ligands. Replacement of the VIP-Thr(11) by an Arg(11) in these ligands contributed to their selectivity: Arg(11)-VIP had a 200-fold lower affinity when compared with VIP at VPAC(2) receptors as opposed to 3- to 5-fold higher affinity at VPAC(1) receptors. Comparison of the binding and functional properties of related VIP analogues suggested that the VPAC(1) selectivity of Arg(11)-VIP was due to the loss of a hydrogen bond between the hydroxy group of Thr residue and the VPAC(2) receptor, steric hindrance between the Arg side chain and the VPAC(2) receptor and charge attraction by the VPAC(1) receptor. Comparison of the ability of VIP analogues to activate adenylate cyclase through chimaeric VPAC(1)/VPAC(2) and VPAC(2)/VPAC(1) receptors indicated that the first extracellular receptor loop carried most of the VPAC(2) receptors' ability to discriminate VIP from Arg(11)-VIP. Based on results obtained for a truncated VPAC(2) receptor and the closely related PACAP-preferring receptor (PAC(1)) and secretin receptors, we hypothesized that Thr(11) interacted with the VPAC(2) receptor Tyr(184) (similar to the VPAC(1) receptor Phe(200) residue). The Y184F (Tyr(184)-->Phe) VPAC(2) mutant lost the ability to discriminate VIP from Val(11)-VIP, and the F200Y VPAC(1) mutant acquired the ability to discriminate the natural peptide from Val(11)-VIP. These results support the hypothesis that the hydroxy group of the native VIP-Thr(11) side chain can indeed form a hydrogen bond with the Tyr side chain in the VPAC(2) receptor.