Elsevier

Cellular Signalling

Volume 17, Issue 1, January 2005, Pages 17-24
Cellular Signalling

Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC1 receptor impair VIP-stimulated [Ca2+]i increase but not adenylate cyclase stimulation

https://doi.org/10.1016/j.cellsig.2004.05.009Get rights and content

Abstract

The vasoactive intestinal polypeptide (VIP) VPAC1 receptor is preferentially coupled to Gαs protein that stimulates adenylate cyclase activity and also to Gαq and Gαi proteins that stimulate the inositol phosphate/calcium pathway. Previous studies indicated the importance of the third intracellular loop of the receptor for G protein coupling. By site-directed mutation of the human recombinant receptor expressed in Chinese hamster ovary cells, we identified two domains in this loop that contain clusters of basic residues conserved in most of the G-protein-coupled seven transmembrane domains receptors. We found that mutations in the proximal domain (K322) reduced the capability of VIP to increase adenylate cyclase activity without any change in the calcium response, whereas mutations in the distal part of the loop (R338, L339, R341) markedly reduced the calcium increase and Gαi coupling but only weakly the adenylate cyclase activity. Thus, the interaction of different G proteins with the VPAC1 receptor involves different receptor sub-domains.

Introduction

Vasoactive intestinal polypeptide (VIP) is a neuropeptide that contributes to the regulation of intestinal secretion and motility, of exocrine and endocrine secretions [1], and to homeostasis of the immune system [2]. VIP's effects are mediated through interaction with two receptor subclasses named VPAC1 and VPAC2 receptors [3]. These receptors are members of the GPCR-B family that also includes the receptors for PACAP, secretin, glucagon, GLP-1, calcitonin, parathyroid hormone and GRF. These receptors are preferentially coupled to Gαs protein that stimulates adenylate cyclase activity and induces cyclic AMP increase [3]. The coupling of the VPAC1 receptor to the inositol phosphate (IP3)/calcium pathway was also demonstrated in cell lines expressing the recombinant VPAC1 receptor [4] and cell lines expressing constitutively the receptors [5], [6]. The VPAC1 receptor-mediated IP3 and calcium increase is partially blocked by pertussis toxin pretreatment attesting a contribution of both Gαq and Gαi proteins [7].

When numerous studies were performed to precise the receptor's domains involved in ligand recognition and receptor activation, little is known about the molecular mechanism involved in G protein recognition and activation. The study of a large panel of chimeric and mutated receptors of the rhodopsin family indicated that the second (IC2), the third (IC3) intracellular loops and, to a lesser extent, the proximal part of the C-terminal tail were directly involved in G protein/receptor interactions [8]. However, the diversity of sequences and loop sizes, even among related receptors, has made difficult the identification of a specific set of sequences (if any exists) defining the coupling profile. A recent study from Couvineau et al. [9] identified in human recombinant VPAC1 receptor two charged amino acids required for adenylate cyclase activation: a lysine located in IC3 (K322) and a glutamic acid located in the proximal C-terminal intracellular tail (E394). On the other hand, we have recently shown that the replacement of a short sequence in the IC3 loop of the VPAC1 receptor by its counterpart in the VPAC2 receptor markedly reduced the calcium response except that mediated by the Gα16 protein [7]. The aim of the present work was to investigate the contribution of other parts of IC3 in the activation of the calcium effector. For that purpose, we constructed mutants by substituting by alanine single or groups of amino acids conserved among the GPCR-B family: the K322–L323–R324 and the R338–L339–A340–R341 sequences located in IC3 loop but also E394 located in proximal C-terminal tail and identified by Couvineau et al. as essential for a normal adenylate cyclase activation. The present results confirm that the two K322 and E394 residues previously identified are indeed involved in adenylate cyclase activation, establish that they are not essential for VIP-mediated calcium increase and identify the R338–L339–A340–R341 sequence located at junction of IC3 and TM6 as necessary for coupling to the IP3/calcium pathway through Gαi.

Section snippets

Construction and expression of VPAC1 wild-type and mutant receptors

The Chinese hamster ovary (CHO) cell line expressing human VPAC1 receptor has been detailed in previous publication [4].

Generation of mutant receptors was achieved using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla CA, USA) as described [7]. Identification of the correct mutation was achieved by DNA sequencing on an ABI automated sequencing apparatus, using the BigDye Terminator Sequencing Prism Kit from ABI (Perkin-Elmer, California, USA). Following DNA sequencing of the

Point mutation in amino-domain of IC3 loop and carboxy-tail of VPAC1 receptor

In a first set of experiments, we evaluated the consequences of single mutations in the conserved proximal part of the VPAC1 receptor IC3 loop (Fig. 1) and of mutation of the E394 residue in the proximal part of the intracellular carboxy-terminus. The mutants K322A, L323A, R324A and E394A were expressed at the membrane and were functional. As previously described for the wild-type receptor, VIP-stimulated cAMP and [Ca2+]i increases are correlated with receptor density up to 5 pmol/mg prot. [7].

Discussion

The current model of receptor activation proposes that agonist induces conformational changes moving TM3 near TM5/6 allowing the receptor to switch from inactive to active state. TM proximity is accompanied by intracellular domain movements that allow efficient coupling to G protein [8]. Residues located in the IC3 loop at junction with transmembrane domains have been proposed to be directly involved in G protein/receptor coupling, for instance, in muscarinic [13], [14], [15], adrenergic [16],

Acknowledgements

This work was financially supported by Interuniversity Attraction Poles Programme P5/20 (Federal Public Planning Service Science Policy). We thank Mélanie Van Craenenbroeck, Ingrid Nachtergael and Christelle Langlet for their help in the realization of some experiments.

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