Conformational switches in the VPAC₁ receptor

Abstract

The vasoactive intestinal peptide receptor 1 (VPAC₁) belongs to family B of GPCRs and is activated upon binding of vasoactive intestinal peptide (VIP) and pituitary AC-activating polypeptide neuropeptides. Widely distributed throughout body, VPAC₁ plays important regulatory roles in human physiology and physiopathology. Like most members of the GPCR-B family, VPAC₁ receptor is predicted to follow the actual paradigm of a common ‘two-domain’ model of natural ligand action. However the precise structural basis for ligand binding, receptor activation and signal transduction are still incompletely understood due in part to the absence of X-ray crystal structure of the whole receptor and to significant structural differences with the most extensively studied family of receptor, the GPCR-A/rhodopsin family. Here, we try to summarize the current knowledge of the molecular mechanisms involved in VPAC₁ receptor activation and signal transduction. This includes search for amino acids involved in the two-step process of VIP binding, in the stabilization of VPAC₁ inactive and active conformations, and in binding and activation of G proteins.

LINKED ARTICLES

This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1

The vasoactive intestinal peptide receptor 1 (VPAC₁) is a member of the family B of GPCRs, which includes VPAC₂, pituitary AC-activating polypeptide receptor 1 (PAC₁), secretin, glucagon, glucagon-like peptide (GLP) 1 and 2, calcitonin, gastric inhibitory polypeptide (GIP), corticotropin-releasing factor (CRF) 1 and 2, and parathyroid hormone (PTH) receptors. The endogenous ligands of VPAC₁ receptor are vasoactive intestinal polypeptide (VIP) and pituitary AC-activating polypeptide (PACAP), two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and to homeostasis of the immune system (Dickson and Finlayson, 2009). Like all members of the GPCR-B family, VPAC₁ receptor is preferentially coupled to Gαs protein that stimulates AC activity and induces cyclic AMP increase, although a coupling to the PLC and the calcium/inositol trisphosphate pathway through either Gαq or Gαi is also effective (Dickson and Finlayson, 2009). VPAC₁ receptor was also reported to interact with receptor activity-modifying proteins (RAMP), in particular RAMP2, inducing a significant increase of agonist-induced inositol trisphosphate production without modifying cAMP stimulation (Christopoulos et al., 2003). Like most GPCRs, VPAC₁ receptor also forms constitutive homodimers as well as hetero-oligomers with VPAC₂ receptors, as demonstrated using biophysical methods (Harikumar et al., 2006), but the physiological consequences of those oligomerizations remain to be elucidated. Indeed, pharmacological studies performed on CHO cells co-expressing VPAC₁ and VPAC₂ receptors did not identify any differences in VIP or selective agonist affinities or potencies. Similarly, VIP receptors co-expression did not modify receptor internalization and trafficking patterns following agonist exposure (Langer et al., 2006).

Major advances in structural biology of GPCRs came a few years ago from solving the first X-ray crystal structures of rhodopsin and ligand-activated GPCR-A family members bound to an antagonist and an agonist (Palczewski et al., 2000; Cherezov et al., 2007; Rasmussen et al., 2007; Jaakola et al., 2008; Park et al., 2008; Scheerer et al., 2008; Warne et al., 2008; Rosenbaum et al., 2011; Xu et al., 2011). However, the mechanisms regulating the GPCR-B family signal transduction are less precisely understood, since no X-ray crystal structure of the whole receptor is available, and conserved motifs of the GPCR-A family (E/DRY at TM3, NPXXY at TM7) are absent in the GPCR-B family. They also differ from family A members by their larger binding site located both on N-terminal extracellular domain and transmembrane (TM) helices. Although recent studies have solved the structure of the N-terminus of several family B receptors (CRF, PTH, PAC₁, GIP, GLP-1, calcitonin receptor-like/RAMP1) and clarified their role in ligand binding (Grace et al., 2007; Parthier et al., 2007; Sun et al., 2007; Pioszak and Xu, 2008; Runge et al., 2008; ter Haar et al., 2010), information on the events that follow ligand binding only came from site-directed mutagenesis and pharmacological studies. These will be developed in this review, trying to highlight the current knowledge of the molecular switches driving VPAC₁ from inactive to active conformation and subsequent G protein binding and activation.

The ‘two-domain’ model for ligand-receptor interaction

The commonly accepted model for agonist action of family B GPCRs suggests that the N-terminal domain of the receptor is the principal binding site for the central and the C-terminal regions of the natural ligand and ensures correct ligand positioning, whereas binding of residues 1–6 of the ligand to the extracellular loops and TM helices drives the receptor activation (Hoare, 2005). Following agonist binding, subsequent conformational changes are expected within the TM domains of the receptor causing key sequences located in the intracellular loops to be exposed and to interact with the G proteins. More recently, it has also been proposed that a helix N-capping motif, identified in the N-terminus of all GPCR-B family ligands and stabilizing their helical conformation, was probably formed upon receptor binding and could also constitute a key element in receptor activation (Neumann et al., 2008).

A large number of site-directed mutagenesis studies suggests that VIP–VPAC₁ receptor interaction also follows this paradigm and pointed out that the N-terminus of the VPAC₁ receptor plays a key role in agonist binding (Laburthe et al., 2007). Solano et al. (2001) also found, using reciprocal substitution mutants in both ligand and receptor, that D³ of VIP forms a salt bridge with R¹⁸⁸ of the VPAC₁ receptor and that this interaction was necessary for receptor activation. More recently, photoaffinity experiments performed by the group of Couvineau and Laburthe showed that benzophenone-residues in position 6, 22, 24 and 28 of VIP are in direct contact with D¹⁰⁷, G¹¹⁶, C¹²² and K¹²⁷ respectively, four residues located in the N-terminus of VPAC₁ receptor (Couvineau et al., 2010). Interestingly, they also observed, using a VIP and a VPAC₁ antagonist affinity probe in position 0, that the N-terminal domain of VIP (agonist) and of the VPAC₁ antagonist recognizes two different microdomains in the N-terminus of the VPAC₁ receptor, while the central and the C-terminal regions of these ligands seem to share the same binding site (Ceraudo et al., 2008a) (Figures 1 and 2).

Figure 1.

Snake plot representation of VPAC₁ receptor. Amino acid sequence of human VPAC₁ receptor, the position of signal peptide, glycosylated residues and amino acids important for VIP binding, receptor activation and G protein coupling are also labelled.

Figure 2.

Amino acid sequence of VIP. Amino acids that were experimentally mapped into the VPAC₁ receptor binding site are in bold and those involved in the helical N-cap are underlined.

Molecular mechanisms involved in VPAC₁ receptor activation

As mentioned before, the recent solving of the X-ray crystal structures of several GPCR-A family members provides clues to the TM helix rearrangements that result from agonist binding and subsequent receptor activation. These include the disruption of an ionic interaction between the cytoplasmic face of TM3 (E/DRY motif) and TM6 (E residue) maintaining the receptor preferentially in a ground inactive conformation in absence of agonist (ionic lock), a ‘rotamer toggle switch’ (modulation of the helix conformation around a proline-kink) in TM6 causing key sequences to be exposed to cytoplasmic binding partners and a conformational change of Y residue of the NPXXY motif located in TM7 stabilizing the active conformation (Rosenbaum et al., 2009; Rosenbaum et al., 2011). In the absence of X-ray crystal structure of the VPAC₁ receptor, only model structures have been reported, which used as template the structures of the N-terminal domain of the CRF 2β receptor (Ceraudo et al., 2008b) or structures of family A GPCRs for the TM domains (Conner et al., 2005; Chugunov et al., 2010). However, the low sequence identity between the VPAC₁ receptor sequence and the templates used for homology modelling prevents direct transposition of molecular switches that drive GPCR-A members activation.

As all members of GPCR-B family, VPAC₁ receptor lacks the E/DRY sequence. On the basis of subtle changes observed when Y²³⁹ and L²⁴⁰, located in TM3 of VPAC₁, were substituted with alanine it was proposed that this YL sequence was equivalent to the E/DRY motif of GPCR-A family (Tams et al., 2001). Another model based on a three-dimensional analysis of the GLP-1 receptor proposed that an E/DRY motif could be formed by three non-adjacent residues consisting in R¹⁷⁴ in the cytoplasmic end of TM2, E²³⁶ and Y²³⁹ in the distal part of TM3 of VPAC₁ (Frimurer and Bywater, 1999). But in our hands Y²³⁹A, L²⁴⁰A, E²³⁶A, Y²³⁹A and R¹⁷⁴A mutants were undistinguishable from the wild-type receptor (Nachtergael et al., 2006). One possible explanation for the discrepancy can be the fact that Tams et al. (2000) studied cyclic AMP measurements in intact cells a more sensitive model than the AC assay on membrane used in our study. Nevertheless, even if the YL motif of GPCR-B family and E/DRY motif of GPCR-A family have the same location, they certainly do not have the same importance for receptor activation (Figure 1).

More recently, by combining pharmacological and in silico approaches, we have identified a network of interactions between residues located in helices 2, 3 and 7 of the VPAC₁ receptor, which are involved in the stabilization of the receptor in the absence of agonist and in early steps of receptor activation. We proposed that, in the absence of ligand, interaction between R¹⁸⁸, N²²⁹ and Q³⁸⁰ ties helices 2, 3 and 7 together (Figure 3). Upon VIP binding, the interaction between R¹⁸⁸ and N³⁸⁰ is broken, and a stronger interaction (salt bridge) is established between R¹⁸⁸ and the D³ side chain of VIP. TM2 and, probably, other helices undergo conformational changes causing key sequences located in intracellular loops to be exposed and to interact with the G proteins. In the meantime, the interaction network involving N²²⁹ and Q³⁸⁰ maintains TM7 in a conformation necessary for proper activation of G proteins. The three-dimensional model also suggested that Q³⁸⁰ could function as a floating ‘ferry-boat’, switching between R¹⁸⁸ and N²²⁹ residues' side-chains, hence contributing to signal transduction propagation and activation of G proteins (Chugunov et al., 2010). Likewise, other studies have pointed out the importance of TM2 and TM7 in G protein activation. Indeed, the mutation into arginine of H¹⁷⁸ located at the bottom of TM2 led to a constitutively activated VPAC₁ receptor (Gaudin et al., 1998). On the other hand, it has also been shown that E³⁹⁴ located at the junction of TM7 and the C-terminus of VPAC₁ was important for VIP-induced cAMP production but was not directly involved in Gαs binding (Couvineau et al., 2003; Langer and Robberecht, 2005). Moreover, we found that phosphorylation levels and internalization of N²²⁹A and N²²⁹Q VPAC₁ receptors (mutants that failed to generate the G protein active state and, therefore, to activate AC properly and to stimulate intracellular calcium increase but with a preserved affinity for VIP and sensitivity to GTP) were comparable with that of the wild-type receptor (Nachtergael et al., 2006). These later results thus suggest that receptor conformation necessary for activation and regulatory mechanisms, such as desensitization and internalization, could be different.

Figure 3.

Three-dimensional model of the TM domains of the VPAC₁ receptor. Lateral (left) and top (right) view of a working model of human VPAC1 receptor, TM and residues identified as important for receptor stabilization are also labelled. Details regarding modelling procedure are described in Chugunov et al. (2010).

When considering other site-directed mutagenesis studies, it is likely that a complex and larger network of interaction between TM helices must be considered for stabilization of VPAC₁ inactive and active conformations (Figure 1). Indeed, mutation of T³⁴³, located at the junction of the third intracellular loop and TM6 of VPAC₁, into lysine, proline or alanine also led to a constitutively activated receptor (Gaudin et al., 1999). Another study showed that Y¹⁴⁶ and Y¹⁵⁰, located in TM1 of VPAC₁, do not interact directly with VIP but stabilize the correct active receptor conformation (Perret et al., 2002). Similarly, we observed that K¹⁹⁵ and D¹⁹⁶ located at junction of TM2 and the first extracellular loop were essential for VPAC₁ activation but were not directly involved in VIP recognition (Langer et al., 2003).

How all these residues cooperate to propagate signal transduction after VIP binding remains to be elucidated and would require a model of the activated receptor in complex with VIP. Particularly the two N-terminal residues of VIP, H¹ and S², are likely to affect, directly or indirectly, the interaction network surrounding N²²⁹ and Q³⁸⁰. Of interest, so far as all residues that were identified as important for VPAC₁ receptor activation are highly conserved among GPCR-B family members, they may, therefore, be involved in a binding and activation mechanism that is common to the whole family.

Molecular mechanisms involved in VPAC₁/G protein binding and activation

The α subunit of heterotrimeric G proteins has a central role in interaction with both the receptor and the effectors. Several studies have shown that the C-terminal part of Gα subunit can directly bind to the receptor and is involved in the coupling specificity (Conklin et al., 1996). The current model of GPCR activation, based on the study of family A GPCRs, proposes that when the receptor switches to its active conformation, TM movements are accompanied by intracellular loops switches leading to exposure of the G protein-binding pocket to cytosol and efficient binding to G protein. However, the diversity of sequences and loop sizes, as well as their flexibility, has made difficult the identification of a specific set of residues defining the coupling profile.

For the VPAC₁ receptor, Gα binding domains are mainly located in the third intracellular loop (IC3), which contains subdomains dedicated to the recognition of the different Gα subunits (Figure 1). K³²² located in proximal part of IC3 and E³⁹⁴ located at the junction of TM7 and the C-terminal tail are required for AC activation but not for the coupling to the inositol trisphosphate/calcium pathway. The former being involved in direct interaction with Gαs (G protein binding), as demonstrated by a reduced sensitivity to GTP, while E³⁹⁴ triggering switch of Gαs from inactive to active state (G protein activation) (Couvineau et al., 2003; Langer and Robberecht, 2005). Similarly, two other sequences located in IC3 have been identified as important for VIP-induced intracellular calcium increase but not cAMP production. A small sequence, I³²⁸-R³²⁹- K³³⁰-S³³¹, located in the central part of IC3 is involved in efficient binding of VPAC₁ to Gαi/o and Gαq (Langer et al., 2002), while R³³⁸ and L³³⁹, located at the distal part of IC3, mediate interaction of VPAC₁ with Gαi/o (Langer and Robberecht, 2005). Combining mutations in the proximal and distal part of IC3 together with mutation of E³⁹⁴ gave rise to a completely inactive VPAC₁ receptor with respect to AC activation and intracellular calcium increase.

Among the different members of the GPCR-B family, proximal and distal domains of IC3 share conserved sequences that could therefore represent common G protein binding motifs. In line with this hypothesis, studies performed on other members of the GPCR-B family identified the proximal domain of IC3 as essential for AC activation but the amino acids involved may differ and additional conserved sequences located in other intracellular regions of the receptor may also be necessary as seen for glucagon (IC2) (Cypess et al., 1999) and calcitonin gene-related peptide receptors (R¹⁵¹ located in IC1) (Conner et al., 2006). The junctions of IC3 loop are predicted to be α-helical and it is assumed that the correct positioning of charged amino acids plays an important role in G protein interaction. However, other data suggest that lipophilic and aromatic residues are also important for G protein interaction. It is possible that IC3 loop junctions activate G protein directly or that they may serve as regions that control the loop conformation. As mutations may change both direct interaction site and secondary structure, it is difficult to define more precisely the mechanisms involved in IC3 loop/G protein interaction. Again a structure or a model of the activated VPAC₁ receptor in complex with VIP could help to answer this question.

Conclusion

Identification of the precise molecular mechanisms that drive GPCRs from inactive to active state represents a major focus in functional genomics and drug development research with the ultimate aim of designing molecules able to stabilize one of these states. VIP and PACAP receptors have been identified as potential therapeutic targets for metabolic, inflammatory and neuronal diseases (Dickson and Finlayson, 2009). But the use of their natural ligands is limited by their lack of specificity (PACAP binds with high affinity VPAC₁, VPAC₂ and PAC₁ receptors while VIP recognizes both VPAC₁ and VPAC₂ receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptide small molecules or specific stabilized peptidic ligands is of high interest. Up to now, only two small molecules antagonists of VPAC₂ receptor have been identified by high-throughput screening (Chu et al., 2010), further investigation and new insight toward elucidation of VIP receptors activation mechanism would allow the rational design of potential new drugs.

Glossary

IC

intracellular loop

PACAP

pituitary AC-activating polypeptide

TM

transmembrane

VIP

Vasoactive Intestinal Peptide

Conflict of interest

The author has no conflicts of interest and no financial links with manufacturers of reagents relevant to this work.