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Constitutive Activation of the HumanVIP1 Receptor

PASCALE GAUDIN, CHRISTIANE ROUYER-FESSARD, ALAIN

COUVINEAU, JEAN-JOSE MAORET, AND MARC LABURTHEa

Laboratoire de Neuroendocrinologie et Biologie Cellulaire Digestives,Institut National de la Santé et de la Recherche Médicale, INSERMU410, Faculté de Médecine Xavier Bichat, B.P. 416, 75870 Paris Cedex18, France

During the past few years a subfamily of the superfamily of G protein–coupled receptorshas emerged that shares the seven membrane-spanning domain topography but has a

low overall amino acid sequence homology (<20%) with other members of the superfam-ily.1,2 This subfamily, now referred to as the class II G protein–coupled receptor family, com-prises receptors for a family of structurally related peptides that includes vasoactive intestinalpeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon,secretin, glucagon-like peptide (GLP-1), gastric inhibitory polypeptide (GIP), growthhormone-releasing peptide (GRF), and, more unexpectedly, also comprises receptors forparathyroid hormone (PTH) and calcitonin.1,2 Recent studies have extended this subfamily1

with the discovery of subtypes of the abovementioned receptors as well as two new membershaving an extraordinarily long N-terminal domain: the putative EGF module-containing,mucin-like hormone receptor EMR1 and the leukocyte activation antigen CD97.

Constitutively active mutants of several G protein–coupled receptors have been char-acterized experimentally by site-directed mutagenesis3 and have been also described aspathogenic in humans.4,5 A constitutively active mutant of a class II G protein receptor hasbeen reported for the PTH-PTH-related peptide receptor in Jansen-type metaphyseal chon-drodysplasia that affects a strictly conserved histidine residue in the first intracellular loopof this class of receptor.6

In this context, we investigated the constitutive activation of the human VIP1 receptor, aclass II G protein–coupled receptor whose structure-function relationship has been previouslydocumented.1 In this paper, we demonstrate that specific mutation of histidine 178 in the VIPreceptor causes its constitutive activation and show that constitutive activation requires theintegrity of the natural ligand binding site in the N-terminal extracellular domain.

RESULTS AND CONCLUSION

Histidine (H) 178 has been mutated into arginine (mutant H178R) by site-directedmutagenesis and the mutated cDNA transfected into Cos cells for subsequent intracellularcAMP measurement. The basal cAMP level was 3.5-fold higher in cells transfected withH178R than the basal cAMP level measured after transfection of the wild type humanVIP1 receptor (TABLE 1) or the vector alone (not shown). This latter observation suggests

aCorresponding author: Tel.: 33 01 44 85 61 35; Fax: 33 01 44 85 61 24; E-mail: laburthe@bichat.inserm.fr

that the wild type receptor was not itself constitutively activated. The cAMP response inCos cells transfected by H178R mutant and wild type receptors was tested upon stimula-tion by VIP. Maximal cAMP responses were identical and half-maximal stimulationsabove basal level were obtained for similar concentrations of VIP in cells transfected withthe H178R receptor or the wild type receptor, i.e., 1.5±0.5 × 10-10 M and 0.4±0.1 × 10-10 M,respectively (not shown). Scatchard analysis of competitive inhibition of 125I-VIP bindingto Cos cell membranes by native VIP indicated that the H178R receptor mutant bound VIPwith a similar dissociation constant as compared to the wild type receptor (TABLE 1). Inthese experiments the concentration of VIP binding sites was higher in cells transfectedwith the wild type receptor than in cells transfected with the H178R receptor mutant(TABLE 1). This observation did not favor the hypothesis that the higher basal cAMP levelin cells transfected with H178R could be merely related to a higher expression of thereceptor as compared to cells transfected with the wild type receptor inasmuch as the wildtype receptor is not constitutively activated by itself (see above). However, in order to doc-ument this issue, we transfected Cos cells with increasing concentrations of cDNA encod-ing the H178R mutant or the wild type receptor. For the receptor mutant, we observed thatthe more cDNA transfected the more cAMP level in Cos cells (not shown). In sharp con-trast, the basal cAMP level in Cos cells transfected with the wild type receptor was con-stant regardless of the amount of cDNA transfected. This latter observation further arguedagainst constitutive activity of the wild type human VIP1 receptor itself.

Further experiments were carried out to determine whether mutation of H178 intoresidues other than arginine also resulted in constitutive activation of the human VIP1receptor. We mutated H178 into a neutral residue (H178A), an acidic one (H178D), or abasic one (H178K). Among these mutants, none was constitutively activated after transfec-tion in Cos cells (TABLE 1). Moreover, these mutants were unable to mediate VIP-stimulatedcAMP production (not shown) likely because they no longer bound VIP (TABLE 1). In orderto determine the pattern of expression of these inactive mutants in transfected Cos cells,immunofluorescence studies were performed after insertion of a Flag at the intracellular C-terminus of the mutated and wild type receptors as previously described.7 Data indicatedthat the mutants H178A, H178D, and H178K, which no longer bound VIP, exibited thesame pattern of expression as the wild type receptor and the mutant H178R (not shown). As

GAUDIN et al.: VIP RECEPTOR CONSTITUTIVE ACTIVATION 383

TABLE 1. Basal cyclic AMP level and VIP binding parameters of wild type and mutatedhuman VIP1 receptors expressed in Cos cells

Constructs cAMP Binding ParametersBasal Level Dissociation Constant Binding Capacity

(pmoles/106 cells) (nM) (pmoles/mg of protein)

wt 18.8 ± 3.7 0.6 ± 0.1 6.1 ± 1.5H178R 67.4 ± 9.9* 0.2 ± 0.1 2.0 ± 0.9H178A 25.8 ± 9.5 ND NDH178D 18.3 ± 5.3 ND NDH178K 13.4 ± 4.6 ND NDH178R-D68A 20.1 ± 5.5 ND NDD68A 16.4 ± 5.3 ND NDH178R-E36A 22.2 ± 2.6 ND NDE36A 18.8 ± 2.6 ND NDH178R-D132A 52.6 ± 4.4* 1.1 ± 0.6 3.0 ± 0.6D132A 22.2 ± 4.8 0.7 ± 0.1 1.6 ± 0.3

Note: Cyclic AMP levels were measured by radioimmunoassay as described.7 Binding parameterswere determined by Scatchard analysis of competitive inhibition of specific 125I-VIP binding byincreasing concentrations of native VIP.7 ND, not detectable; wt, wild type. Results are mean ± SEof at least three experiments. * Values significantly different, p < 0.01, from that of wt.

expected for proteins in an active phase of synthesis in transfected cells,7,8 immunofluores-cence could be detected at the plasma membrane and also in intracellular compartments.

That constitutive activation of the human VIP1 receptor by mutation of H178 into argi-nine did not happen with mutations into alanine, aspartate, and even lysine and that suchmutations also abolished VIP binding suggested that such constitutive activation might bedependent on the integrity of the VIP binding site. In order to investigate this issue we con-structed double mutants in which the mutation resulting in the constitutive activation of thereceptor (H178R) was associated with point mutations in the N-terminal extracellulardomain of the receptor, which abolished VIP binding as shown, i.e., D68A9 or E3610. Asexpected the H178R-D68A mutant no longer bound VIP like the single mutant D68A(TABLE 1). Nor did it mediate the stimulation of cAMP production by VIP (not shown).TABLE 1 further shows that this double mutant is no longer constitutively activated whenexpressed in Cos cells. Like the single mutant E36A, the H178R-E36A mutant no longerbound VIP (TABLE 1) and did not mediate the stimulation of cAMP production by VIP (notshown). The H178R-E36A double mutant was not constitutively activated when expressedin Cos cells (TABLE 1). As a control, we developed another double mutant in which H178Rwas associated with a point mutation (D132A) in the N-terminal extracellular domainwhich was previously shown not to alter VIP binding.10 As expected, the H178R-D132Amutant, like the single mutant D132A, bound VIP with a dissociation constant similar tothat of the wild type receptor (TABLE 1). This mutant also mediated VIP-stimulated cAMPproduction (not shown). Interestingly the double mutant H178R-D132A did exhibit con-stitutive activation upon transfection in Cos cells (TABLE 1).

Constitutive activation evoked by replacement of histidine 178 by arginine in thehuman VIP1 receptor is much less efficient than activation induced by the natural agonistVIP in the H178R mutant receptor or the wild type receptor. Nevertheless, the construc-tion of double mutants supports a close relationship between the ligand-dependent activa-tion triggered by VIP and the ligand-independent one evoked by mutation of histidine 178into arginine. Indeed, our original approach consisting of constructing double mutants sug-gests that the conformational change triggering constitutive activation in the H178R recep-tor mutant may transit over the ligand binding site in the N-terminal extracellular domainand requires the structural and functional integrity of this domain. This double mutationapproach, which invalidates the agonist binding domain, was feasible for the VIP receptorbecause the binding site in the N-terminal extracellular domain1 is well separated from thesite of the mutation evoking constitutive activation.

Since histidine 178 in the human VIP1 receptor is strictly conserved in all members of theclass II family of receptors, it may be of general functional importance in these G protein-coupled receptors. Whether mutation of the equivalent histidine in other peptide receptor ofthis family results in ligand-independent activation has been only reported for the PTHreceptor.6 In this context, it is worth pointing out that two members of this family are recep-tors having an extraordinary long N-terminal extracellular domain with unique features: theputative EGF module-containing, mucin-like hormone receptor EMR111 and the leukocyteactivation antigen CD97.12 Since the signaling pathway(s) are not known, experimentalmutation of the equivalent histidine in these two receptors could be instrumental in demon-strating their possible coupling with adenylyl cyclase.

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