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Structure. Author manuscript; available in PMC Jul 1, 2009.
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PMCID: PMC2601695
NIHMSID: NIHMS59812

This article has been retractedRetraction in: Structure. 2012 June 2; 19(8): 1200    See also: PMC Retraction Policy

Structure of the parathyroid hormone receptor C-terminus bound to the G-protein dimer Gβ1γ2

Summary

A critical role of the Gβγ dimer in heterotrimeric G-protein signaling is to facilitate engagement and activation of the Gα subunit by cell-surface G protein-coupled receptors. However, high-resolution structural information of the connectivity between receptor and Gβγ has not previously been available. Here, we describe the structural determinants of Gβ1γ2 in complex with a C-terminal region of the parathyroid hormone receptor-1 (PTH1R) as obtained by x-ray crystallography. The structure reveals that several critical residues within PTH1R contact solely Gβ residues located within the outer edge of WD1 and WD7 repeat segments of the Gβ toroid structure. These regions encompass a predicted membrane-facing region of Gβ thought to be oriented in a fashion that is accessible to the membrane-spanning receptor. Mutation of key receptor contact residues on Gβ1 lead to a selective loss-of-function in receptor/heterotrimer coupling while preserving Gβ1γ2 activation of the effector phospholipase-C beta.

Introduction

Heterotrimeric G-proteins, composed of Gα, Gβ, and Gγ subunits, transmit information from extracellular cues to a vast array of intracellular signal transduction cascades and thereby regulate a variety of cellular functions (McCudden et al., 2005; Wettschureck and Offermanns, 2005). Heterotrimeric G-proteins are activated by cell surface-spanning, G protein-coupled receptors (GPCRs), which catalyze exchange of GTP for GDP on Gα to initiate signal propagation. While activated, GTP-bound Gα regulates a number of downstream effectors and governs signal duration by possessing the GTPase activity required for signal termination; the Gβγ subunit freed from Gα also modulates many signaling proteins (McCudden et al., 2005; Wettschureck and Offermanns, 2005). Additionally, the Gβγ subunit is indispensable to receptor-mediated activation of heterotrimeric G-proteins (Fung, 1983). Increasing evidence also suggests a direct role of the Gβγ subunit in the activation process per se (Johnston and Siderovski, 2007b; Johnston et al., 2005; Rondard et al., 2001; Van Eps et al., 2006) leading to a model in which receptor uses direct contacts between its intracellular loops and Gβγ to tilt Gβγ relative to Gα, which subsequently ‘levers’ open a feasible route for the release of GDP from Gα. Moreover, a sequential-fit model has been proposed for the coupling of Gαβγ to receptor that suggests receptor/Gβγ contacts govern the primary and necessary event leading to subsequent receptor/Gα binding and Gα activation (Herrmann et al., 2004). However, precise molecular determinants for Gαβγ coupling to receptor as well as the mechanism of receptor-mediated activation of heterotrimer remain poorly defined and thus subject to much speculation (reviewed in (Johnston and Siderovski, 2007a)).

We recently determined the structure of Gαi1 bound to a specific region of the third intracellular (ic3) loop of the D2-dopamine receptor, providing the first high-resolution structural determinants of a receptor/Gα contact site and highlighting a role of the Gα β6 strand in nucleotide exchange (Johnston and Siderovski, 2007b). Several studies have reported that Gβγ also interacts directly with the ic3 loop of certain receptors (Mahon et al., 2006; Taylor et al., 1996; Wu et al., 2000). As the ic3 loop also interacts with Gα, a coincident interaction with Gβγ could possibly occur through separate protomers of a receptor dimer (Johnston and Siderovski, 2007a). Unfortunately, many studies investigating the Gβγ/ic3 loop interaction have used receptors with large ic3 regions (~100 amino-acids), affording limited detail on precise residues involved in binding Gβγ (Wu et al., 1998; Wu et al., 2000). More recently, however, a report investigating the interaction of Gβγ with the parathyroid hormone receptor-1 (PTH1R) described a minimal interaction domain within its C-terminal region essential for PTH1R-mediated signaling through several Gα subfamilies, suggesting that diverse Gα subunits can use a common receptor/Gβγ interaction as the basis for receptor coupling and activation (Mahon et al., 2006).

To gain insight into the structural determinants of this receptor/Gβγ connectivity, we solved the crystal structure of Gβ1γ2 bound a C-terminal region of the parathyroid hormone receptor-1 (PTH1R). The structure has highlighted a specific receptor/Gβ contact surface that involves the WD1 and WD7 repeat segments of Gβ1, both of which are predicted membrane-proximal regions previously implicated in receptor coupling.

Results

High affinity interaction between Gβ1γ2 and the PTH1R C-terminus

We first confirmed the high-affinity Gβγ/PTH1 receptor C-terminal tail interaction (Mahon et al., 2006) by using quantitative surface plasmon resonance (SPR) binding analysis. Biotinylated PTH1R C-tail peptide was coupled to a streptavidin SPR sensor; injection of purified, recombinant Gβ1γ2 dimer over this surface resulted in a specific, robust interaction with an apparent dissociation constant (KD) of 109 ± 4 nM (Figure 1), confirming a direct interaction between Gβ1γ2 and the C-terminal tail of the PTH1 receptor. The high affinity nature of this interaction supports a proposed model suggesting that a receptor/Gβγ interaction represents the initial step in proper G-protein coupling to receptor (Herrmann et al., 2004).

Figure 1
Direct binding of Gβ1γ2 to the PTH1R C-terminal tail peptide. Purified Gβ1γ2 protein at indicated concentrations was injected over a streptavidin SPR biosensor surface, previously coated with biotin-PTH1R peptide (aa 466-487), ...

Crystal structure of the Gβ1γ2/PTH1R C-tail complex

We next determined the molecular basis for this interaction using x-ray crystallography. A complete diffraction pattern dataset was collected on a single crystal containing a complex between non-lipidated Gβ1γ2 and the PTH1R C-tail peptide and refined to 3.0 Å Bragg diffraction (Table 1). The overall structure of the complex (Figure 2A) reveals an 8-residue span of the receptor C-tail (W474-L481) bound to the “top” of the Gβ toroid and engaging residues within the WD1 and WD7 repeats. This surface of Gβ is predicted to face the plasma membrane in the proposed receptor-bound conformation of the heterotrimer, an orientation that would allow access to the intracellular surface of a membrane-bound GPCR (Hamm, 2001). Previous studies have implicated this general region of Gβ in the interaction of Gβγ with receptors (Hou et al., 2001; Taylor et al., 1996; Wu et al., 1998). Notably, no PTH1R peptide contacts were identified with the Gγ subunit. Suitable electron density was found for Gγ extending to arginine-62, which is five amino acids short of the isoprenylated CAAX-box at the Gγ C-terminus (68CAIL71 in wildtype Gγ2; 68SAIL71 in the CAAX-box mutant Gγ2(C68S) used in this study). Previous reports have indicated that direct receptor contacts with the C-terminus of Gγ are critical to receptor/G-protein signaling and may confer a level of specificity to the interaction as well as participate in the nucleotide exchange process induced by receptor/Gβγ interactions (Azpiazu and Gautam, 2001; Hou et al., 2000; Kisselev and Downs, 2003). Given our Gβ1γ2/PTH1R peptide structure and its lack of Gγ engagement with receptor, these previously described receptor/Gγ interaction(s) likely involve a region of the receptor distinct from the sequence encompassed by the PTH1R peptide.

Figure 2
Crystal structure of the Gβ1γ2 / PTH1R peptide complex (PDB id 2QNS). (A) Overall structure represented in two angular depictions, with Gβ1γ2 dimer colored green and orange, and the ordered residues of the PTH1R C-tail ...
Table 1
X-ray data collection and refinement statistics for PDB entry 2QNS

Most notable of the specific contacts made between Gβ and the PTH1R C-tail peptide (Figures 2B and and3)3) are two tryptophan residues: Trp-474 of PTH1R makes a probable π-cation interaction with Arg-52 of Gβ1 and additional interactions with Arg-49 and Thr-50 of Gβ1; Trp-477 of PTH1R makes several van der Waals contacts with Trp-339 and Lys-337 of Gβ1 and additional interactions with Thr-47 and Gly-310 of Gβ1. Our results are congruent with the original finding of this Gβγ/PTH1R interaction which also reported the critical involvement of these two tryptophan residues (Mahon et al., 2006). Additional PTH1R residues making key Gβ contacts are Thr-478 and Leu-481 (Figures 2B and and33).

Figure 3
Mapping of PTH1R C-terminal tail contacts onto the Gβ1 primary and secondary structure. (A) Primary sequence alignment and secondary structure features of the human Gβ subunit family comprising Gβ1 through Gβ5. Secondary ...

Validation of the Gβ1γ2/PTH1R interface by mutagenesis and functional studies

To confirm the binding interface seen in the crystal structure, specifically with respect to the involvement of Trp-474 and Trp-477 (Figure 4A), we tested the ability of Gβ1γ2 to bind a predicted loss-of-function, alanine-mutated peptide: W474A/W477A. Gβ1γ2 binding to this alanine-mutated peptide was dramatically reduced compared to wild-type peptide (Figure 4B), confirming the critical nature of these residues to Gβγ binding.

Figure 4
Validation of the Gβ1γ2 / PTH1R peptide interface by PTH1R mutational analysis. (A) Tryptophans W474 and W477 of PTH1R (transparent space-filling spheres) are predicted to make critical Gβ1 contacts, with W477 packing extensively ...

Mutation of these two critical tryptophan residues that contact Gβ1 was previously shown to abolish PTH-stimulated inositol phosphate accumulation (Mahon et al., 2006). To further validate the model of the Gβ1γ2/PTH1R contact surface, we also performed whole-cell inositol phosphate accumulation assays to assess PTH1R-mediated activation of phospholipase C via Gβγ. These experiments employed a Gβ1 construct in which three residues of the PTH1R binding interface were mutated to alanine: Gβ1 residues T47, K337, and W339 (Figure 5A). This triple-alanine Gβ1 mutant is expressed well in cellular co-transfection with Gγ2, but unable to interact with biotinylated PTH1R C-tail peptide in pulldown experiments (Figure 5B), in contrast to wildtype Gβ1. Cells co-transfected with expression vectors for full-length PTH1R and wild-type Gβ1γ2 showed enhanced response to the PTH1R agonist, PTH(1-34) peptide, compared to cells transfected with receptor cDNA alone (Figure 6A). This enhanced agonist response likely occurs due to the transfected Gβ1γ2 increasing the pool of G-protein heterotrimer available to activated receptor. In contrast, cells co-transfected with PTH1R and alanine-mutated Gβ1γ2(TKW>A) failed to elicit a response above that of receptor alone (Figure 6A), suggesting that these three mutations within Gβ1 indeed abolish proper receptor coupling and activation. This lack of a Gβ1γ2(TKW>A)-mediated increase was not due to inability of the mutant Gβγ to stimulate inositol phosphate production, as the Gβ1γ2(TKW>A) dimer was equally competent as wildtype to stimulate overexpressed PLC-β2 under agonist-independent conditions (Figure 6B), confirming that the alanine mutations do not compromise the effector-binding site of Gβγ which is typically centralized to the pore region of the Gβ toroid as described by several crystal structures and mutagenesis studies (Figure 7). Overall, these results suggest that the Gβ1 surface contacting the PTH1R C-tail peptide in the Gβ1γ2/PTH1R crystal structure is important for PTH1 receptor coupling to Gβγ.

Figure 5
Examination of the Gβ1γ2 / PTH1R peptide interface by Gβ1 mutational analysis. (A) Electron density representation of the final Gβ1γ2/PTH1R dimer model highlighting Gβ1 residues Thr-47, Lys-337, and Trp-339. ...
Figure 6
Validation of the Gβ1γ2 / PTH1-receptor interface by Gβ1 mutational analysis and cellular assessment of receptor coupling to effector activation. (A) COS-7 cells were transfected with human PTH1 receptor cDNA and either empty pcDNA3.1 ...
Figure 7
The interaction points of multiple Gβγ-binding proteins mapped onto the surface representation of the Gβγ dimer. In all panels (A-I), the Gβ1γ2 structure (PDB entry 1TBG) is depicted in surface-filling representation ...

Discussion

Of the nine Gβ1 residues contacting the PTH1R peptide (Figure 3), eight are identical and one is conserved (Lys-337 in Gβ1-3 vs Arg-337 in Gβ4) among Gβ1-4 isoforms. Thus, we speculate that the Gβ1/PTH1R interface herein described may represent a contact site for additional GPCRs among these four Gβ subunits. Receptor-specific contacts may be afforded by additional Gβ- as well as Gγ-binding sites (Azpiazu and Gautam, 2001; Hou et al., 2001). Comparing all Gβ1-5 isoforms reveals less sequence identity in PTH1R contact points (5 of 9), including a particular non-conservative change (Gln-44 in Gβ1-4 vs Val-44 in Gβ5) rendering Gβ5 less likely to support hydrogen bonds from Thr-478 and Leu-481 at this position. Moreover, the Gβ5 subunit has been previously characterized as a non-canonical Gβ which dimerizes with GGL domains of RGS proteins rather than traditional, isolated Gγ subunits (Sondek and Siderovski, 2001). In SPR experiments similar to those of Figure 1, a purified Gβ5/RGS9 dimer (Cheever et al., 2008) failed to interact with the PTH1 receptor peptide (data not shown). These results, however, do not exclude an interaction of the Gβ5/RGS9 dimer with a similar region of other receptors and/or distinct region(s) of specific receptors.

Our previous results with the dopamine D2-receptor ic3 loop peptide provided the first high-resolution glimpse of the molecular determinants for a receptor/Gα contact site critical for G-protein activation (Johnston and Siderovski, 2007b). The present Gβ1γ2/PTH1R structure now adds a parallel high-resolution description of the structural basis of a receptor/Gβγ contact site. As the structure of a complete GPCR/Gαβγ complex remains elusive, these individual ‘snapshots’ together help shape our understanding of how receptors directly engage heterotrimeric G-proteins. Indeed, in model renderings of the receptor/Gαβγ complex based in part on our current findings (Figure 8), a monomeric GPCR is seen to lack the ability to simultaneously accommodate both the Gαi1/D2N and Gβ1γ2/PTH1R interactions, whereas a dimeric GPCR is seen to accommodate both structural constraints. Although receptor dimerization is considered by many to be a crucial step in receptor-mediated G-protein activation (George et al., 2002; Prinster et al., 2005), several recent studies have indicated that a monomeric GPCR can serve as the minimal functional unit for efficient G-protein activation (Bayburt et al., 2007; Ernst et al., 2007; White et al., 2007; Whorton et al., 2007). Interestingly, the particular study by Bayburt and colleagues demonstrated that isolated receptor dimers can only couple efficiently to a single G-protein heterotrimer (Bayburt et al., 2007). Thus, while a monomeric receptor can stimulate nucleotide exchange, the overall receptor/Gαβγ interaction may be more multifaceted. Further pursuit of additional receptor/Gα and receptor/Gβγ complexes will undoubtedly help complete and refine our current view of the receptor/Gαβγ complex.

Figure 8
Predicted interaction of the heterotrimeric G-protein with a receptor monomer and dimer. The Gαi1β1γ2 structure (PDB entry 1GP2) is depicted with Gα colored green, Gβ blue, and Gγ cyan. The bound GDP molecule ...

One prevailing model of receptor-mediated G-protein activation evokes a direct role of Gβγ in suggesting that receptors use the Gβγ subunit as a ‘lever’ to create a GDP exit route from the Gα subunit (Johnston and Siderovski, 2007b; Johnston et al., 2005; Rondard et al., 2001). We speculate that the Gβ1γ2/PTH1R interaction may represent a direct contact site exploited by activated receptor to induce such allosteric conformational changes at the Gα/Gβγ interface (e.g. the β3/α2 loop) that allow for enhanced GDP release. Our results with the Gβ1(TKW>A) mutant, which retains PLCβ activating function but lacks the ability to couple receptor to its proper agonist-mediated response, are suggestive of this scenario placing Gβγ connectivity with the receptor C-tail directly in the path of receptor-mediated G-protein activation. However, we cannot yet exclude the alternative possibility that the lack of response observed with the Gβ1(TKW>A) mutant is due to improper receptor coupling with heterotrimer, irrespective of the actual nucleotide exchange event. Nevertheless, the Gβ1γ2/PTH1R C-tail interaction described herein undoubtedly represents a critical site for heterotrimeric G-protein coupling to the PTH1 receptor and, perhaps, serves as a site of action through which the agonist-activated receptor ultimately achieves guanine nucleotide exchange.

Experimental Procedures

Materials

Rat PTH1R C-terminal peptides (466VQAEIRKSWSRWTLALDFKRKA487), with and without N-terminal biotinylation, were separately synthesized by Fmoc-protection and purified via HPLC by the Tufts University Core Facility; the negative control biotinylated peptide mNotch has previously been described (Snow et al., 2002). Expression vectors for myc-tagged, wildtype Gβ1 and HA-tagged Gγ2 were obtained as previously described (Snow et al., 1999); the TKW>A triple-mutant of Gβ1 was generated by Quickchange PCR-based site-directed mutagenesis (Stratagene, La Jolla, CA) and sequence verified before use. Unless otherwise noted, all other materials were purchased from Sigma Aldrich (St. Louis, MO).

1γ2 purification

High 5 insect cells (Invitrogen), at a density of ~2 × 106 cells/mL, were co-infected with high-titer baculoviruses encoding human Gβ1 and Gγ2 (the latter with a hexahistidine N-terminus). An additional modification was made to the Gγ2 subunit by replacing the cysteine at residue 68 with serine (C68S). This mutation renders the well-characterized CAIL (CAAX) motif necessary for isoprenylation as SAIL, incapable of undergoing this modification and thus resulting in expression of a cytosolic Gβγ protein complex. Infections were carried out at 27°C for 50 hr. Cells were harvested by centrifugation at ~2000×g for 20 min at 4°C. Cell pellets were resuspended in N1 buffer (20 mM HEPES pH 8.0, 300 mM NaCl, 1 mM EDTA, 5% glycerol, 20 mM imidazole, 1% cholate, protease inhibitor cocktail), lysed in an Avestin Emulsiflex, and clarified by ultracentrifugation (45,000×g for 1 hr). Although the C68S-modified Gβ1γ2 dimer is not predicted to be membrane tethered, we routinely include 1% cholate in the initial N1 buffer to help prevent non-specific protein adherence to the NiNTA column, yielding higher purity following this initial chromatography step. Gβ1γ2 dimer was then purified using sequential Ni-NTA affinity, ion-exchange, and gel exclusion chromatographies using previously described techniques (Johnston et al., 2005). Protein was concentrated to 9 mg/ml in 20 mM HEPES pH 8.0, 1 mM EDTA, 2 mM DTT, and 50 mM NaCl using YM-30 Centricon concentrators (Millipore).

Surface plasmon resonance

SPR analyses were carried out at 25 °C on a BIAcore 3000. N-terminally biotinylated peptides (diluted to 0.5 μg/ml in BIA buffer [10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.005 % NP-40]) were coupled to separate flow cells of a streptavidin biosensor (Biacore) to surface densities of ~1000 RU. Gβ1γ2 (30 μL) was then injected simultaneously over flow cells at 10 μL/min followed by 300 sec dissociation phase. Binding to a non-Gβγ interacting peptide (biotin-mNOTCH; (Johnston et al., 2005)) was subtracted from all binding curves to correct for nonspecific binding and bulk buffer shifts. Surfaces were regenerated following each Gβ1γ2 injection with a single 10 μL pulse of 0.5 M NaCl / 0.025 M NaOH at 20 μL/min. Kinetic analyses were made using BIAevaluation v3.0. Dissociation constants (KD values) were calculated from sensorgrams using the simultaneous association (ka) and dissociation (kd) analysis parameter.

Structure determination

Initial crystals of Gβ1γ2 in complex with the PTH1R peptide were obtained by vapor diffusion in condition #39 of Crystal Screen I (Hampton Research). Optimized crystals were obtained in hanging drops (7 μL) containing a 1:1 (v/v) ratio of protein (9 mg/ml Gβ1γ2 with 2-fold molar excess of PTH1R peptide) and well solution (1.9 M ammonium sulfate, 2.5% PEG-400, 0.1 M HEPES pH 6.8-7.0). Crystals formed within 7 days and grew for 2-3 weeks to maximum dimensions (~250 × 50 × 50 μm). For data collection at 100K, a single crystal was transferred to cryoprotectant buffer (well solution supplemented with 15% glycerol) for 30 sec prior to immersion in liquid nitrogen. A native dataset was collected at UNC using a Rigaku R-Axis-IV++ detector with rotating anode generator and Osmic Blue confocal optics. Diffraction data were scaled and indexed using HKL2000 (Otwinowski, 1993). Crystals belong to the space group I222 with cell dimensions of a = 63.26 Å, b = 112.92 Å, c = 141.48 Å and α = β = γ = 90°. Although the crystal diffracted to ~2.4 Å, data was manually truncated to 3.0 Å due to unusually high Rmerge values at higher resolution data bins. The Gβ1γ2/PTH1R structure (PDB id 2QNS) was solved by molecular replacement (AmoRe; (Navaza, 1994)) using the structure of Gβ1γ2 (PDB id 1GP2) with all non-protein atoms and Gαi1 removed. Model building and refinement (including real space, rigid body, simulated annealing, energy minimization, and b-factor protocols) was carried out using Coot (Emsley and Cowtan, 2004) and CNS (Brunger et al., 1998). Unless otherwise noted, all structural images were made using PyMol (DeLano Scientific, San Francisco).

Cellular assays

For Gβ1γ2 pulldown assays, COS-7 cells were grown in 6-well dishes and transfected with pcDNA3.1-based DNA expression vectors encoding wildtype or mutated Myc-tagged Gβ1 (or empty vector control) plus HA-tagged Gγ2 in a 1:1 ratio (total 1.5 μg DNA plus 4.5 μL Fugene-6 per well). After 48 hours, two wells per condition were lysed by sonication in 2.1 mL of lysis buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 0.01% SDS, and Complete protease inhibitors [Roche]). Samples were then centrifuged for 10 minutes at 16,300 × g before the top 1 mL of the supernatant was removed and incubated with 2.5 μg of either biotinylated-PTH1R C-tail or -mNotch control peptide overnight at 4 °C. 120 μg of streptavidin-agarose beads (Sigma; pre-blocked with 50 μg/mL BSA in lysis buffer) were then incubated with the lysate/peptide mixture for 1 hour prior to being applied to a Micro Bio-Spin chromatography column (BioRad) and washed 4 times with lysis buffer before elution in 2.5× Laemmli protein sample buffer. Precipitated samples, and aliquots of lysate input, were resolved on SDS-PAGE for anti-myc immunoblotting using previously described techniques (Snow et al., 1999). For inositol phosphate accumulation assays, COS-7 cells were grown in 12-well dishes and transfected with expression vectors encoding human PTH1 receptor, Myc-tagged Gβ1, and HA-tagged Gγ2 in a 1:2:2 ratio (total 1 μg DNA plus 3 μL Fugene-6 per well). To examine exogenous PLC-β2 stimulation, cells were transfected with 200 ng of human PLC-β2 expression vector. After 24 hours, cells were treated with [3H]inositol (~1 μCi per well) in serum- and inositol-free DMEM and incubated overnight. Cells were then treated with the PTH(1-34) peptide agonist (100 nM) in the presence of LiCl (10 mM) for 15 minutes at 37 °C. Accumulation of [3H]inositol phosphates was then analyzed as described (Hains et al., 2006).

Acknowledgments

We thank J. Sondek and M. Cheever for Gβ1 and Gγ2 baculoviridae and Gβ5/RGS9 protein, D. Bosch for critical appraisal of the manuscript, and grant support by NIH NRSA F32 GM076944 (to C.A.J.) and NIH R01 GM074268 (to D.P.S.).

Footnotes

Accession Numbers: Atomic coordinates and structure factors for Gβ1γ2 in complex with the PTH1R C-tail peptide have been deposited in the Protein Data Bank under accession code 2QNS.

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