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Molecular Endocrinology, doi:10.1210/me.2005-0169
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Molecular Endocrinology 20 (1): 136-146
Copyright © 2006 by The Endocrine Society

A Docking Site for G Protein ß{gamma} Subunits on the Parathyroid Hormone 1 Receptor Supports Signaling through Multiple Pathways

Matthew J. Mahon, Tabetha M. Bonacci, Paola Divieti and Alan V. Smrcka

Endocrine Unit, Massachusetts General Hospital (M.J.M., P.D.), Boston, Massachusetts 02114; and Department of Pharmacology and Physiology (T.M.B., A.V.S.), University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Address all correspondence and requests for reprints to: Matthew J. Mahon, Endocrine Unit, Massachusetts General Hospital, Wellman 501, 50 Blossom Street, Boston, Massachuesetts 02114. E-mail: mahon{at}helix.mgh.harvard.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The G protein-coupled receptor for PTH and PTH-related protein (PTH1R) signals via many intracellular pathways. The purpose of this work is to investigate a G protein binding site on an intracellular domain of the PTH1R. The carboxy-terminal, cytoplasmic tail of the PTH1R fused to glutathione-S-transferase interacts with Gi/o proteins in vitro. All three subunits of the heterotrimer interact with the receptor C-tail. Activation of the heterotrimeric complex with GTP{gamma}S has no effect on Gß{gamma} interactions, but markedly disrupts binding of the G{alpha}i/o subunits to the receptor tail, suggesting that direct Gß{gamma} binding indirectly links G{alpha} subunits to this region of the receptor. Gß{gamma} subunits alone bind the C-tail with an affinity that is comparable to the heterotrimeric G protein complex. G protein complexes consisting of G{alpha}shis6-ß1{gamma}2 and G{alpha}qhis6-ß1{gamma}2 also interact with the PTH1R tail in vitro. The Gß{gamma} interaction domain is located on the juxta-membrane region of the tail between amino acids 468 and 491. Mutations that disrupt Gß{gamma} interactions block PTH signaling via phospholipase Cß/[Ca2+]i and MAPK and markedly reduce signaling via adenylyl cyclase/cAMP. Herein, we define a domain on the PTH1R that is capable of binding G protein heterotrimeric complexes via direct Gß{gamma} interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PTH 1 RECEPTOR (PTH1R) is a class b G protein-coupled receptor (GPCR). The PTH1R has several distinguishing characteristics. First, the PTH1R mediates the actions of two functionally distinct factors, PTH and PTHrP. PTH is an endocrine factor responsible for maintaining mineral ion homeostasis by targeting bone and kidney. PTHrP, however, is an autocrine/paracrine factor that is critical for the development of many tissues, such as bone and the cardiovascular system (1). For example, PTH1R-null mice die in utero due to defects in cardiovascular development (2). Due to the lack of PTH1R isoforms, the diverse and complex array of actions elicited by PTH and PTHrP presumably occurs via one receptor target.

Perhaps to compensate for the lack of receptor isoforms, activation of the PTH1R is linked to many intracellular pathways. The PTH1R signals primarily via adenylyl cyclase (AC)/cAMP and phospholipase Cß (PLC)/[Ca2+]i through coupling to Gs and Gq/11, respectively (3, 4, 5). Several lines of evidence also link Gi/o activation to the PTH1R based on the effects of pertussis toxin, including the regulation of phosphate transport (6), the activation of PLC (7), increases in [Ca2+]i (8) and suppression of cAMP accumulation (9, 10, 11, 12, 13). The PTH1R also couples to the activation of Ca2+ channels (14, 15), phospholipases D (16, 17), and A2 (18), the MAPK pathway via epidermal growth factor receptor (19), and the release of nitric oxide (20) and the endothelial-derived hyperpolarizing factor (21).

Combined, the PTH1R is capable of coupling to many subclasses of G proteins, including Gs, Gq, Gi/o, and recently coupling to G11 (22) and G12 and G13 (23) have been described. Thus, the PTH1R is part of a growing list of GPCRs that promiscuously couple to multiple G proteins (24). Structure-function studies of the PTH1R revealed that the second and third intracellular loops contain determinants for G protein coupling. Replacing the EKKY amino acid sequence located at the C terminus of the second intracellular loop of the PTH1R with the amino acid sequence DSEL blocks PTH-mediated signaling via PLC without affecting signaling via AC (25). N-terminal regions of the PTH1R’s third intracellular loop contain elements required for coupling to both AC and PLC (26). Despite the absence of sequence homology, receptors belonging to the class a subfamily (rhodopsin/adrenergic receptors) also couple G proteins primarily via the second and third intracellular loops (27). Serial truncations of the PTH1R cytoplasmic, carboxy-terminal tail (C-tail) up to amino acid 480 does not adversely affect PTH signaling via AC or PLC but has a modest reduction in receptor expression (28). However, C-tail truncations proximal to amino acid 480 severely impair receptor expression in either transiently transfected COS7 cells or in stable human embryonic kidney (HEK) 293 cell lines, probably as a result of improper folding and membrane insertion (28). Iida-Klein et al. (29) demonstrated that the PTH1R truncated at amino acid 480 fails to respond to pertussis toxin with an increase in the activation of AC when compared with the full-length receptor, suggesting that Gi/o couples to the C-tail.

The N-terminal region of the PTH1R cytoplasmic tail contains conserved calmodulin binding domains (30). Fluphenazine, a calmodulin antagonist, enhances PTH signaling via PLC; however, the PTH1R containing mutations that block calmodulin binding signals poorly through PLC, suggesting that another factor(s) binds this region. The metabotrophic glutamate receptors also bind calmodulin through determinants on the cytoplasmic tail (31, 32). Notably, this calmodulin interaction domain overlaps a binding site for Gß{gamma} subunits of the heterotrimeric G ptoteins. These findings led us to examine whether the calmodulin interaction domain on the PTH1R also binds to Gß{gamma} subunits. Herein, we describe a Gß{gamma} interaction domain located within the juxtamembrane region of the cytoplasmic tail of the PTH1R. In vitro, direct Gß{gamma} binding to this domain indirectly links G{alpha} subunits of Gi/o, Gs, and Gq subclasses, thus establishing possible mechanisms by which the PTH1R promiscuously couples to many G protein subtypes and intracellular signaling pathways.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTH1R C-Tail and G Protein Interactions
The Gß subunit within a preparation G proteins isolated from bovine brain containing a mixture of G{alpha}i/o and Gß{gamma} subunits displays a robust, specific interaction with the PTH1R C-tail fused to GST (Fig. 1AGo). The specific Gß isoform that interacts with the C-tail is not known because the antibody used to detect the interaction recognizes all isoforms. Estimates suggest that the C-tail/G protein interaction is high affinity (Fig. 1BGo, top panel). G{alpha}i (Fig. 1BGo, lower panel) and G{gamma} (data not shown) subunits also interact with the C-tail, suggesting that the Gi/o proteins bind as a heterotrimeric complex.



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  		Fig. 1. G{alpha}iß{gamma} Interacts with the PTH1R Cytoplasmic Tail (C-Tail) in Vitro

A, GST alone or GST fused to the PTH1R C-tail (~2 µg), partially purified from E. coli extracts and bound to glutathione-Sepharose, were interacted with a bovine brain fraction of G proteins containing Gi/o (40 nM). Interactors were eluted with glutathione and analyzed with pan-specific Gß antibodies (left panel) and the membrane stained with coomassie blue (right panel). B, GST-C-tail was interacted with decreasing amounts of G{alpha}iß{gamma}, as indicated. Interactors were eluted and probed with Gß antibodies (upper panel). The membrane was partially stripped and probed with G{alpha}i antibodies (lower panel). The lower bands on the lower panel are partially stripped antibodies bound to Gß shown in the upper panel.

 
Increasing concentrations of guanosine 5'[{gamma}-thio] triphosphate (GTP{gamma}S) in the presence of 3 mM MgCl2 has no effect on the Gß binding to the PTH1R C-tail up to 100 µM (Fig. 2AGo, upper panel). However, treatment with 100 µM GTP{gamma}S markedly inhibits G{alpha}i binding to the C-tail (Fig. 2AGo, lower panel). These findings suggest two possibilities: GTP{gamma}S-induced conformational changes in the G{alpha} subunit disrupt direct interactions with the C-tail, and/or direct Gß{gamma} binding to the C-tail indirectly links the G{alpha} subunit to this domain, an interaction that is lost upon GTP{gamma}S-induced dissociation of the heterotrimeric G protein complex. Gß1{gamma}2 purified to homogeneity from baculavirus infected Sf-9 cells interacts with the PTH1R C-tail that is qualitatively indistinguishable from the interactions displayed by the Gi/o heterotrimeric complexes (Fig. 2BGo). These findings reveal that Gß and/or G{gamma} directly bind the C-tail and that G{alpha} subunits are not required.



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  		Fig. 2. Gß{gamma} Binds Directly to the PTH1R C-Tail

A, GST-C-tail, expressed and partially purified from E. coli, was interacted with bovine brain G proteins containing G{alpha}i/oß{gamma} (40 nM) in the presence of increasing concentrations of GTP{gamma}S (1–100 µM), as indicated and described in Materials and Methods. Interactors were eluted and analyzed with Gß (upper panel) and G{alpha}i (lower panel) antibodies. B, GST or GST-C-tail were interacted with ß1{gamma}2 expressed and purified from baculavirus infected Sf9 cells at the concentrations indicated. Eluted proteins were analyzed with Gß antibodies. C, GST or GST-C-tail were interacted with the mixture of G{alpha}i/oß{gamma} (40 nM) proteins in the absence or presence of 100 µM GTP{gamma}S, as indicated. Interactors were analyzed with antibodies to Gß (first panel), G{alpha}i-1 (second panel), G{alpha}i-2 (third panel) and G{alpha}o (fourth panel). The faint upper bands in the G{alpha}i-1 panel are cross reactivity with the GST-C-tail protein.

 
Next, the ability of the PTH1R C-tail to pull-down specific G{alpha} subunits was investigated. The G protein preparation from bovine brain contains a mixture of G{alpha}i-1, G{alpha}i-2, G{alpha}i-3, and G{alpha}o. As shown in Fig. 2CGo, G{alpha}i-1, G{alpha}i-2, and G{alpha}o pull-down with the PTH1R C-tail, interactions that are disrupted by GTP{gamma}S treatment. The faint bands above the G{alpha}i-1 bands are nonspecific binding of the antibodies to the microgram amounts of the GST-C-tail protein on the membrane blot. At this stage, it is difficult to ascertain the relative binding affinities among these complexes because the initial concentrations of the specific G{alpha} combinations are unknown and the efficacy of the isoform-specific antibodies appears to differ substantially. Considering that the PTH1R primarily couples to Gs and Gq, we next sought to investigate whether these G{alpha} subunits interact with the C-tail.

To generate G{alpha}s- and G{alpha}q-containing complexes, we chose to use adenoviral-mediated expression of these constituents in a fibroblast cell line. To validate this methodology, PS120 fibroblasts were triply transduced with adenoviruses expressing G{alpha}i-1, his6-Gß1, and G{gamma}2. These G proteins were partially purified using immobilized metal affinity chromatography, as shown in Fig. 3AGo. This G protein preparation avidly binds to the PTH1R C-tail in vitro (Fig. 3BGo), demonstrating that the his6-tag on the Gß subunit does not impair binding. Using the same methodology, G protein preparations enriched with G{alpha}s and G{alpha}q were prepared. As shown in Fig. 3CGo, G{alpha}s and G{alpha}q readily cointeract with Gß and the PTH1R C-tail in vitro. However, unlike G{alpha}i/o subunits, GTP{gamma}S only weakly inhibits G{alpha}s interactions with the C-tail and has no apparent effect on G{alpha}q binding (Fig. 3DGo).



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  		Fig. 3. G{alpha}i-1his6-ß1{gamma}127, G{alpha}shis6-ß1{gamma} G{alpha}qhis6-ß1{gamma} Interact with PTH1R C-Tail in Vitro

A, His6Gß1, G{gamma}2, and G{alpha}i-1 were packaged into adenoviruses, expressed in PS120 fibroblasts and purified using IMAC, as described in Materials and Methods. The partially purified G-protein complex was immunoblotted with Gß (lane 1), G{gamma}2 (lane 2) and G{alpha}i-1 (lane 3) antibodies. B, GST or GST-C-tail were interacted with the IMAC purified G{alpha}i-1-his6ß1-{gamma}2 preparation (~20 nM) and interactors analyzed with Gß (left panel) and G{alpha}i-1 (right panel) antibodies. C, Following the same protocol above, G protein complexes enriched with G{alpha}shis6-ß1{gamma}2 G{alpha}qhis6-ß1{gamma}2 complexes (~20 nM) were mixed with GST or GST-C-tail and interactors analyzed with antibodies to Gß (upper panel), G{alpha}s (lower left panel) and G{alpha}q (lower right panel), as indicated. D, GST-C-tail was interacted with Gi/o preparation (40 nM) from bovine brain (lanes 1 and 2) and the his6Gß1{gamma}2 complexes (~20 nM) enriched with G{alpha}i-1, G{alpha}s, or G{alpha}q, in the absence or presence of 100 µM GTP{gamma}S, as indicated. Interactors were analyzed with antibodies to Gß (upper panel) and the appropriate primary antibodies to the indicated G{alpha} subunit isoforms (lower panels).

 
Defining the G Protein Interaction Domain on the PTH1R C-Tail
Next, the location of the G protein-binding domain on the PTH1R C-tail was determined. As shown in Fig. 4Go, both N- and C-terminal deletions of the C-tail demonstrate that this interaction domain is an extended motif located on N-terminal portions of this cytoplasmic domain, existing between amino acids 468–491. In the context of the full-length receptor, this interaction domain is just C-terminal to the seventh transmembrane domain of the PTH1R. The R485stop mutation, which deletes 106 amino acids from the C terminus, has recently been linked to Eiken syndrome, a rare disorder of the bone (33). This mutation disrupts interactions with Gß{gamma} (Fig. 4DGo), revealing a possible mechanism for the dysplasia associated with the syndrome. Single alanine point mutations between amino acids 473 and 477, which is the region between N{Delta}15 and N{Delta}20 (Fig. 4AGo), fails to disrupt G protein binding (data not shown). To develop point mutations that block G protein binding, an alternate approach was undertaken.



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  		Fig. 4. G{alpha}i-3ß{gamma} Interacts with N-Terminal Portions of the PTH1R C-Tail

A, A schematic depicting N-terminal deletions of the GST-C-tail is shown. The numbers indicate the range of amino acid residues fused to GST. B, GST, GST-C-tail (full), and GST-C-tail with N-terminal deletions of 5 (N{Delta}5), 10 (N{Delta}10), 15 (N{Delta}15) and 20 (N{Delta}20) amino acids were interacted with G{alpha}i/oß{gamma} (40 nM). Interactors were analyzed with Gß (upper panel) and G{alpha}i (middle panel) antibodies. Coomassie blue-stained membrane is shown (lower panel). C, A schematic depicting C-terminal deletions of the GST-C-tail is shown. D, GST, GST-C-tail (full), and GST-C-tail with C-terminal deletions of 85 (C{Delta}85), 95 (C{Delta}95), and 100 (C{Delta}100) amino acids and the 485stop point mutation (R485stop) were interacted with G{alpha}i/oß{gamma} (40 nM). Analysis was as in B.

 
Sequence alignment reveals that this region of the PTH1R is reasonably well conserved among class b GPCRs, especially the tryptophans at positions 473 and 477 (30). The most divergent class b GPCR in this region is the GHRH receptor (GHRHR). Consequently, the GHRHR C-tail does not interact with G proteins in vitro (data not shown). Thus, the GHRHR provides a guide for incorporating mutations that effectively block G protein interactions with the PTH1R C-tail. Two notable differences include a histidine substitution for the tryptophan at position 477 (relative to the PTH1R) and a proline substitution for the phenylalanine at position 483. As shown in Fig. 5AGo, three mutant PTH1R C-tails were developed; the double point mutation consisting of W477H and F483P (HP), a GHRHR/PTH1R chimera (GC) consisting of amino acids 473 to 483 of the PTH1R replaced with the corresponding amino acids from the GHRHR and a double point mutation replacing both conserved tryptophans with alanine (WW; W474A/W477A). These mutations markedly impair Gß and G{alpha}i binding in vitro (Fig. 5BGo). The Gß{gamma} subunits in the absence of G{alpha} display an identical loss of binding to these three mutant receptor tails (Fig. 5CGo).



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  		Fig. 5. Mutational Analysis of the G Protein-PTH1R C-Tail Interaction

A, Sequence alignment of the Gß{gamma} binding site of the PTH1R and corresponding sequences from the GHRHR and sequences of the mutants used in B are shown (mutations are underlined). B and C, GST-C-tail (wild type, WT) and the C-tail containing the following point mutations, W477H/F483P (HP), GHRHR-chimera (GC) and W473A/W477A, were interacted with 40 nM of G{alpha}i/oß{gamma} (B) or Gß1{gamma}2 alone (C). Interactors were analyzed with Gß (upper panels; B and C) and G{alpha}i (middle panel; B only) antibodies. Coomassie blue-stained membranes are shown (lower panels; B and C).

 
The Gß{gamma} Interaction Domain Is Required for PTH Signaling via PLC
Removal of the C-tail in the context of the full-length receptor impairs surface expression to a large degree (28). Incorporating the GHRHR-based mutations into the full-length PTH1R had no effect on surface binding for the PTH1R-HP receptor and only a modest effect on PTH1R-GC (90% of wild type), as determined by PTH(1–34) ligand binding studies (Fig. 6AGo). Incorporating the double tryptophan to alanine mutations (PTH1R-WW) or the Eiken syndrome-associated mutation (R485stop) reduced surface expression to approximately 50% of wild type (Fig. 6AGo), and thus these mutations were not used for the cell-based signaling studies.



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  		Fig. 6. Gß{gamma} Binding Site on the PTH1R Is Required for Signaling via PLC, [Ca2+]i and MAPK in HEK293 Cells

A, Specific binding of radioiodinated PTH(1–34) on HEK293 cells transiently transfected with the wild-type PTH1R (WT), the PTH1R containing the W477H/F483P point mutations (HP), the PTH1R/GHRHR chimera (GC; refer to Fig. 5AGo for sequence), the PTH1R containing the W473A/W477A point mutations (WW) and the PTH1R containing the R485stop mutation (485stop) was determined, (n = 4 ± SEM). Data are representative of three independent experiments. B, HEK293 cells were transiently transfected with PTH1R-WT, PTH1R-HP and PTH1R-GC, labeled with [3H]myo-inositol and treated with 100 nM PTH(1–34) for 15 min. Accumulation of total inositol phosphates was determined. Data are representative of three independent experiments, (n = 6 ± SEM). C, HEK293 cells transfected with PTH1R-WT (dashed line), PTH1R-HP (solid line) and PTH1R-GC (dotted line) were loaded with fura-2 and treated with 100 nM PTH(1–34). Changes in [Ca2+]i were monitored at the single cell level and the traces shown are representative of an average response in a given microscopic field. D, HEK293 cells transfected with PTH1R-WT, PTH1R-HP, and PTH1R-GC were treated with 100 nM PTH(1–34) for the times indicated. Whole-cell extracts (50 µg) were probed with antibodies to phospho-ERK (p-ERK). Immunoblot shown is representative of three independent experiments.

 
The most striking effect of mutating the G protein interaction domain on the C-tail is the loss of PTH signaling via PLC. The PTH1R transiently transfected into HEK293 yields a 3- to 4-fold increase in total inositol phosphates upon stimulation with PTH (Fig. 6BGo). PTH-mediated accumulations of total inositol phosphates in cells expressing either PTH1R-HP (W477H and F483P) or PTH1R-GC (GHRHR chimera), however, are markedly decreased (Fig. 6BGo). The PTH1R expressed in HEK293 cells also displays a robust increase in intracellular calcium ([Ca2+]i), presumably due to an inositol phosphate-mediated release from intracellular stores, as monitored at the single cell level (Fig. 6CGo). Consistent with the inositol phosphate data, PTH-elicited increases of [Ca2+]i mediated by PTH1R-HP and PTH1R-GC were substantially lower than the PTH1R-WT, marked by a lower maximal release followed by a rapid return to baseline levels (Fig. 6CGo). Notably, the PTH1R-HP transfected cells consistently display low, pulsatile fluctuations of [Ca2+]i at an average of two to three peaks with some cells responding up to five times before returning to baseline (data not shown). These data suggest that Gq coupling occurs, probably through the second and third intracellular loops, but to a lesser extent in the absence of the Gß{gamma} binding site on the C-tail.

PTH also signals via the MAPK pathway in transiently transfected HEK293 cells, as determined by a marked increase in phosphorylation of the extracellular regulated kinases (ERKs) p42 and p44 (Fig. 6DGo). This PTH-elicited response peaks at 5 min and persists up to 20 min (Fig. 6DGo). Cells expressing both PTH1R-HP and PTH1R-GC fail to mediate ligand-induced phosphorylation of the ERKs to a similar extent as that displayed by the wild-type receptor with activation of the MAPK pathway by the GC mutant being almost nonexistent (Fig. 6DGo). These data suggest that the G protein interaction domain on the C-tail is required for PTH coupling to the MAPK pathway.

The possibility that these mutations have a global effect and induce improper receptor folding that results in a loss of G protein coupling at sites that are distinct from the C-tail interaction domain delineated in vitro, such as the intracellular loops, exists. To address this possibility, a decoy protein that expresses the G protein interaction domain on the C-tail in the absence of the full-length PTH1R was developed. To stabilize expression of this domain (only 13 kDa), the C-tail was fused to the C terminus of the single-pass transmembrane domain of the PDGFR, as described in Materials and Methods. The extreme C terminus of the PTH1R contains a putative PDZ interaction domain, thus to exclude a potential confounding variable, this domain was removed. This decoy construct is called pHook-CTFC10 and the negative control, which lacks most of the G protein interaction domain, is referred to as pHook-CTN20C10 (Fig. 7AGo). Cotransfection of the wild-type PTH1R and pHook-CTN20C10 into HEK293 cells had no apparent effect on PTH signaling via PLC/inositol phosphates (Fig. 7BGo) or MAPK (Fig. 7CGo). Conversely, equimolar plasmid transfection of the PTH1R and pHook-CTFC10 markedly inhibits PTH-mediated activation of both PLC (Fig. 7BGo) and MAPK pathways (Fig. 7CGo). The C terminus of the G protein receptor kinase 2 (GRK2-CT), a known Gß{gamma} sequestrant, did not inhibit signaling via MAPK (data not shown), suggesting that free Gß{gamma} dimers are not involved in this pathway. Phorbol esters, but not forskolin, robustly activate the MAPK pathway in HEK293 cells, an effect that is not blocked by pHook-CTFC10 transfection (Fig. 7CGo). These data suggest that PTH activates the MAPK pathway via the PLC/protein kinase C pathway in HEK293 cells and that the pHook-C-tail protein effectively squelches signaling by sequestering heterotrimeric G protein complexes.



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  		Fig. 7. The PTH1R C-Tail Functions as a Dominant Negative for PTH Signaling via PLC and MAPK

A, Schematic depicting the pHook-C-tail constructs is shown. The numbers indicate the range of amino acid residues fused to the pHook construct. B, HEK293 cells were transfected with the PTH1R and equal amounts of either pHook-CTN20C10 or pHook-CTFC10, labeled with [3H]myo-inositol and treated with 100 nM PTH(1–34) for 15 min. Accumulation of total inositol phosphates was determined and data are representative of three independent experiments, (n = 6 ± SEM). C, HEK293 cells were transfected with the PTH1R and equal amounts of either pHook-CTN20C10 (left panel) or pHook-CTFC10 (right panel) and treated with 100 nM PTH(1–34) for the times indicated. Whole cell extracts were blotted with pERK antibodies, as before. D, HEK293 cells were transfected with the PTH1R and equal amounts of either pHook-CTN20C10 or pHook-CTFC10, as indicated. Cells were treated with vehicle (DMSO; lanes 1 and 2), PTH(1–34) (100 nM; lanes 3 and 4), forskolin (10 µM; lanes 5 and 6) or PMA (100 nM; lanes 7 and 8) for 5 min, followed by analysis of cell extracts with pERK antibodies.

 
The Gß{gamma} Interaction Domain Supports PTH Signaling via AC
The PTH1R primarily couples to Gs and activation of the AC/cAMP pathway. In the HEK293 model, the PTH1R mediates a 36-fold increase in cAMP upon treatment with 10 nM PTH (Fig. 8AGo). PTH-elicited accumulations of cAMP in cells transfected with PTH1R-HP are comparable to cells transfected with the wild-type receptor (Fig. 8AGo). However, the PTH1R-GC, a more extensive mutation, has a reduced capacity to support signaling via AC/cAMP when compared with the wild-type receptor (Fig. 8AGo). F1–14 cells, an immortalized osteoblast cell line developed from PTH1R-null mice (34), were used to confirm these results in a bone cell line. Adenoviral vectors expressing flag-tagged versions of PTH1R-WT and PTH1R-GC were transduced into F1–14 cells at equal surface expression levels, as determined by cell surface ELISA (see Materials and Methods). As shown in Fig. 8BGo, the maximum accumulation of cAMP generated by the PTH1R-GC is markedly reduced when compared with the PTH1R-WT, suggesting that Gs coupling is impaired.



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  		Fig. 8. Gß{gamma} Binding Site on the PTH1R C-Tail Supports PTH Signaling via AC

A, HEK293 cells were transfected with the wild-type PTH1R (WT), PTH1R-HP (HP), or PTH1R-GC (GC) and treated with either vehicle (10 mM acetic acid) or 10 nM PTH(1–34) for 15 min in the presence of 1 mM 3-isobutyl-1-methylxanthine. Accumulation of cAMP was determined per well. Data are representative of three independent experiments (n = 6 ± SEM). The difference between PTH1R (WT) and PTH1R-GC are statistically significant (P < 0.05). B, PTH1R-null F1–14 osteoblasts were transduced with adenoviruses expressing either flag-tagged PTH1R-WT or flag-tagged PTH1R-GC such that surface expression of both receptors was the same, as determined by a surface ELISA. Cells were treated with PTH(1–34) for 10 min at the concentrations indicated. Data are indicative of three independent experiments (n = 4 ± SEM).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this current work, we identify a novel G protein interaction site on the cytoplasmic, C-terminal tail of the PTH1R just after the seventh transmembrane domain. This domain is rather large comprising approximately 23 amino acids and is amphipathic in nature. Initial structure-function analysis suggests that amino acids containing aromatic side chains (Trp and Phe) in among basic amino acids (Lys and Arg) are key elements of this domain. Pull-down experiments in vitro demonstrate that all three subunits of the heterotrimeric G protein complex interact with the proximal portion of the PTH1R C-tail. Activation induced by GTP{gamma}S disrupts binding of G{alpha}i/o subunits to this domain in vitro without affecting Gß{gamma} binding, suggesting that the Gß{gamma} directly binds this site and that G{alpha} interacts indirectly via direct Gß{gamma} interactions. However, GTP-induced conformational changes disrupting direct G{alpha} binding cannot be ruled out at this stage. Furthermore, it is likely that in the absence of a full-length, ligand-bound receptor the GDP for GTP{gamma}S exchange on Gs and Gq proceeds with slower kinetics, thus reducing subunit dissociation. Perhaps the C-tail contains direct binding sites for G{alpha}s and G{alpha}q, experiments that will require purification of the G{alpha} subunits free of the Gß{gamma} dimer.

As previously mentioned, PTH1R coupling to Gs and Gq/11 is well documented. Point mutations that block Gß{gamma} binding to the N-terminal region of the C-tail block PTH signaling via PLC, intracellular calcium and MAPK. Combined, these data imply that this domain provides a docking site for Gq. PTH signaling via AC/cAMP is markedly reduced only for the PTH1R-GC mutant, suggesting that coupling of Gs is less dependent upon this domain. Mutational analyses demonstrate that both the second (25) and third (26) intracellular loops of the PTH1R contain G protein-coupling determinants. Thus, it is likely that the cytoplasmic domains of the PTH1R collectively form a multifaceted interaction domain for G proteins. The data presented herein suggest that the Gß{gamma} binding domain on the proximal portion the cytoplasmic tail is required for Gq coupling. Coupling of Gs to the PTH1R is only supported by this domain probably due to a higher intrinsic affinity of the G{alpha}s subunit for other cytoplasmic domains, such as the third intracellular loop. Recently, using bioluminescence resonance energy transfer, Gales et al. (35) reported that several GPCRs precouple G proteins before ligand binding. Perhaps the Gß{gamma} interaction domain enhances PTH signaling via Gs by localizing the heterotrimeric complex to the cytoplasmic domains of the PTH1R.

For the most part, the uncoupling of G protein-specific signaling pathways via site-directed mutagenesis has identified G protein coupling domains located on GPCRs. Despite the absence of sequence homology with the PTH1R, the N-terminal regions of the cytoplasmic tails of rhodopsin (36), ß2-adrenergic receptor (27), oxytocin receptor (37), and endothelin receptors (38) are also essential for G protein coupling and activation. Notably, class a receptors contain a helical fourth cytoplasmic loop that is in the same relative location of the PTH1R Gß{gamma} interaction domain (39). For rhodopsin, this eighth helix mediates coupling with transducin (40). Reports demonstrating direct binding between G proteins and GPCRs through specific domains are not as prevalent.

Neubig and co-workers (41, 42) identified a G protein-binding domain by cross-linking a peptide derived from the third intracellular loop of the {alpha}2-adrenergic receptor. This receptor domain directly contacts both G{alpha} and Gß (42). Gß{gamma} dimers directly bind to the third intracellular loops of M2 and M3 muscarinic receptors, an interaction that directs GRK2-dependent phosphorylation, promoting internalization and desensitization (43, 44). Unlike the PTH1R, G{alpha} blocks Gß{gamma} binding to the loops of the muscarinic receptors, suggesting a different binding mechanism.

Analogous to the PTH1R, the metabotrophic glutamate receptors directly bind Gß{gamma} dimers via determinants located within N-terminal portions of the cytoplasmic tail that also indirectly link G{alpha} subunits to the receptor (31, 32). Notably, the Gß{gamma} binding site distinctly overlaps a calmodulin-binding domain. Recently, our laboratory reported a calcium-dependent interaction with calmodulin (30) that binds a site that also overlaps the Gß{gamma} domain described herein. Sequence comparison between the PTH1R and the metabotrophic glutamate receptors in this region, however, does not reveal a distinct motif aside from the presence of hydrophobic and basic amino acid residues. Despite the absence of discernable sequence homology, Gß{gamma} interaction domains have been found on class a ({alpha}2-AR), class b (PTH1R) and class c (metabotrophic glutamate receptors) GPCRs, suggesting that direct Gß{gamma} binding participates in coupling-specific pathways to receptors.

The function of a particular G protein heterotrimer is primarily linked to the G{alpha} subunit. However, a growing body of evidence demonstrates an important role for Gß{gamma} with respect to receptor coupling and activation of downstream effector molecules (45). Using an antisense oligonucleotide approach, Kleuss et al. (46) demonstrated that somatostatin receptor-mediated inhibition of voltage-sensitive calcium channels is dependent upon G{gamma}3 in GH3 rat pituitary cells, whereas muscarinic receptor-mediated inhibition of calcium influx via these same channels is dependent upon G{gamma}4. This seminal work clearly demonstrates a role for specific Gß{gamma} combinations in regulation of receptor signaling. Kisselev and Gautam (36) reported that the {alpha}{tau} subunit of transducin (Gt) readily forms complexes with Gß1{gamma}1, Gß1{gamma}2, and Gß1{gamma}3, but only the Gß1{gamma}1 combination supports {alpha}t coupling to light-activated rhodopsin. Notably, G{gamma}1 is specifically expressed in the rod photoreceptors, thus demonstrating cell-specific expression of a G{gamma} isoform that directs receptor signaling. Robishaw and co-workers (47), using ribozyme-mediated gene suppression, demonstrated that the D1 dopamine receptor uses Gß1{gamma}7 for activation of AC, whereas suppression of Gß1{gamma}7 had no effect on AC activation mediated by the closely related D5 dopamine receptor in HEK293 cells. Using the same experimental paradigm, the Gß1{gamma}7 combination is also required for ß2-AR signaling via AC (48). Receptor-specific Gß{gamma} combinations that support high-affinity, G protein-dependent agonist binding to receptors have been reported for adrenergic receptors (49), adenosine receptors, and the 5-hydroxytryptamine receptor (50). Combined, these data reveal a vital role for Gß{gamma} during agonist recognition of receptor and regulation of signaling and thus lends credence to the importance of the Gß{gamma} interaction domain located on the PTH1R.

One salient feature of this interaction domain is that it is capable of binding inactive heterotrimeric G protein complexes. Studies investigating G protein interactions have focused largely on G{alpha}ß{gamma} subunit interactions and binding to and regulation of effector molecules. One common theme of these studies is that the interaction domains that link G{alpha} to Gß{gamma} overlap domains that mediate interactions with effector molecules (51). Thus, Gß{gamma}-mediated regulation of effectors, such as PLCß2 and ß3, adenylyl cyclase (types I and II), and GIRK and Ca2+-channels, is inhibited by G{alpha} (51, 52). Direct binding of Gß{gamma} to the C terminus of GRK2 also requires G{alpha} dissociation, which results in membrane localization of the kinase and enhanced phosphorylation of activated GPCRs (53, 54). Gß{gamma} binding the PTH1R C-tail, however, is not blocked by G{alpha}, but instead G{alpha} is a component of the receptor-G protein complex, at least in vitro. These findings strongly suggest that the receptor interacts with Gß{gamma} on sites that are distinct from the G{alpha} and effector binding sites.

In conclusion, the cytoplasmic domains of the PTH1R undoubtedly interact with the heterotrimeric G proteins via a multifaceted binding mechanism. Herein, we delineate one of several G protein-binding interfaces on the PTH1R that favors interactions with the Gß{gamma} dimer. Thus, the Gß{gamma} interaction domain on the C-tail likely represents a common binding site for the many G proteins that couple to the PTH1R, thus establishing a possible mechanism that reflects the promiscuous nature of the PTH1R and the complex signaling patterns elicited by PTH and PTHrP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The pGex vector and glutathione-Sepharose were from Amersham Pharmacia Biotech (Uppsala, Sweden). All primary and secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), specifically, G{alpha}s/olf (C-18; sc-383), G{alpha}i-1 (R4; sc-13533), G{alpha}i-2 (T-19, sc-7276), G{alpha}i-3 (C-10; sc-262), which recognizes both G{alpha}i-1 and G{alpha}i-3 and to a lesser extent G{alpha}i-2, G{alpha}o (A2; sc-13532), G{alpha}q (E-17; sc-393), Gß (T-20; sc-378) which recognizes all Gß isoforms, and G{gamma}2 (A-16; sc-374). Pfu polymerase was from Stratagene (La Jolla, CA). DNA restriction enzymes and T4 DNA ligase were either from Promega (Madison, WI) or New England Biolabs (Beverly, MA). cAMP RIA kit, [3H]-myo-inositol and Western Lightning chemiluminescence reagents were from New England Nuclear (Boston, MA). The p30 vector was from Novagen (San Diego, CA). All reagents and vectors for the production of adenoviruses and baculoviruses (Virapower) were from Invitrogen (Carlsbad, CA). Forskolin, phorbol 12-myristate 13-acetate, GTP{gamma}S, and Gi/o proteins from bovine brain were from Calbiochem (San Diego, CA). Fura-2-AM was from Molecular Probes (Carlsbad, CA). All other general chemicals were from either Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

GST Pull-Down Assay
The GST pull-down assay was used as essentially described (8) with some modifications. Briefly, the full-length carboxy-terminal, cytoplasmic tail (C-tail) of the PTH1R, between amino acids 463 to 591, was cloned on the carboxy terminus of GST using the pGEX vector. The full-length PTH1R C-tail is poorly expressed in Escherichia coli due to an apparent toxicity. To reduce leaky expression, the GST-C-tail cassette was cloned into the p30 vector, which places expression under the control of T7 polymerase. Despite these changes, a significant portion of the protein expressed is GST alone most likely due to translational termination.

Amino-terminal amino acid deletions of the C-tail of five (amino acids 468–591), 10 (473–591), 15 (478–591), and 20 (483–591) and caboxy-terminal deletions of 85 (463–506), 95 (463–496), 100 (463–491) and 106 (R485stop; 463–484) were generated using PCR and pfu polymerase. Silent mutations yielding BspEI and BamHI restriction enzyme sites located on the 5' and 3' flanks of the Gß{gamma} interaction domain respectively were incorporated to facilitate development of the point mutations. All point mutations were generated using PCR/pfu as well. The carboxy tail of the GHRHR (amino acids 376–419) was cloned into the modified p30-GST vector. GHRHR amino acids 387–396 were cloned into the PTH1R using the BspEI and BamHI restriction sites on the C-tail to develop the GHRHR/PTH1R chimera. All sequences were verified using an ABI Prism Sequencer.

Glutathione-Sepharose beads were loaded with approximately 2 µg of C-tail protein and mixed with the G protein preparations end-over-end at 4 C in a buffer containing 25 mM HEPES (pH 7.4), 20% glycerol, 100 mM NaCl, 1 mM dithiothreitol, 0.01% Thesit, 0.01% Triton X-100, 10 mg/ml E. coli extracts and a protease inhibitor cocktail (Sigma). In the noted experiments, GTP{gamma}S was added up to 100 µM to the interaction buffer supplemented with 3 mM MgCl2. Additional experiments performed at room temperature and using up to 5 mM MgCl2 displayed no discernable difference on the G protein interaction, especially with G{alpha}s and G{alpha}q. Bead pellets were then washed with 3 x 1 ml of the above interaction buffer lacking the E. coli extracts, followed by elution with 5 mM glutathione and analysis with SDS-PAGE and immunoblotting using enhanced chemiluminescence.

Viral Expression and Purification of G Proteins
All adenoviruses were developed using the Virapower system from Invitrogen, following the manufacturer’s protocols. Briefly, cDNAs for G{alpha}i-1, G{alpha}s, G{alpha}q, his6-Gß1, and G{gamma}2 were cloned in the pENTR vector and recombined with the pAd/CMV/V5-Dest vector using LR Recombinase. These adenoviral vectors were transfected into 293A cells using FuGene 6 (Roche, Indianapolis, IN) to package the virus for use in transduction experiments. The fibroblast cell line, PS120, was used to generate G proteins due to its high proliferation rate and adenoviral transduction efficiency. PS120 cells were triply transduced with equal amounts of the G{alpha}-his6ß1{gamma}2 combinations at an MOI between 10 and 20 to ensure high levels of expression and purified using immobilized metal-affinity chromatography, as described (55) with some modifications. Membranes from approximately 100 million cells were prepared and extracted in 1% sodium cholate, 25 mM HEPES (pH 7.4), 20% glycerol, 50 mM NaCl, 10 µM GDP, and protease inhibitors. Final concentrations of 0.5% Thesit, 250 mM NaCl, and 10 mM imidazole were added to the cholate extracts, followed by loading on to a Ni2+-NTA column (QIAGEN, Valencia, CA). The column was washed with 50 ml of 25 mM HEPES (pH 7.4), 20% glycerol, 250 mM NaCl, 0.5% Thesit, 10 mM imidazole, and 10 µM GDP. G proteins were eluted from the column with wash buffer supplemented with 200 mM imidazole. Eluates were dialyzed against a buffer containing 25 mM HEPES (pH 7.4), 20% glycerol, 50 mM NaCl, 0.1% Thesit and 1 mM dithiothreitol using a slide-a-lyzer cassette (Pierce, Rockford, IL). Protein amounts were estimated by immunoblotting with a pan-specific Gß antibody compared with known amounts of Gß.

Free Gß1{gamma}2 subunits were purified from 2 liters of Sf-9 cells triply infected with his6-G{alpha}i-1, Gß1, and G{gamma}2 essentially as described (55).

Cell-Based Assays and Analysis of PTH Signaling
HEK293 and F1–14 cells were grown in DMEM supplemented with 10% fetal bovine serum and antibiotics. The wild-type PTH1R and the various mutants were transiently transfected into HEK293 cells using FuGene 6. Specific binding of radioiodinated PTH(1–34) was done as previously described (25). Forty-eight hours after transfection, PTH elicited accumulations of cAMP were quantified by RIA (New England Nuclear) and total inositol phosphates were determined as previously described in detail (29). For MAPK pathway signaling, after a time-course of PTH(1–34) treatment whole-cell extracts of transfected HEK293 cells were isolated using a buffer containing 25 mM HEPES (pH 7.4), 20% glycerol, 0.5% Triton X-100, 1 mM EDTA, 10 mM ß-glycerophosphate, 10 mM sodium pyrophosphate, 1 mM sodium fluoride, and a protease inhibitor cocktail. Equal amounts of extracts were loaded on the SDS-PAGE and analyzed by immunoblotting with phospho-specific ERK1/ERK2 antibodies on Tyr-204. Analysis of calcium transients is as essentially described in detail (8) with the following modification. Chambered cover glass units were coated with poly-lysine before seeding HEK293 cells to enhance adherence.

Adenoviruses expressing flag-tagged versions of the PTH1R and PTH1R-GC (GHRHR-chimera) were developed essentially as described above. F1–14 cells are simian virus 40 large T antigen immortalized osteoblasts isolated from PTH1R-null mice (34). F1–14 cells were transduced with varying amounts of adenoviruses expressing either the wild-type flag-PTH1R or flag-PTH1R-GC. Surface expression of the receptors was quantified using a surface ELISA, essentially as described (56). Briefly, 48 h after transduction, the cells were fixed in freshly prepared 2% paraformaldehyde in PBS for 15 min. Cells were washed and blocked in PBS (without detergent) containing 5% nonfat dry milk for 30 min, followed by sequential incubations and washes with the flag-specific antibody (Sigma) and an antimouse secondary antibody labeled with horseradish peroxidase. The horseradish peroxidase substrate o-phenylenediamine at 0.4 mg/ml in 50 mM phosphate-citrate (pH 5.0), and 0.03% sodium perborate was added to the wells for 10–15 min. Reactions were stopped with 3 N HCl and quantified spectrophotometrically at 492 nm. F1–14 cells were transduced with volumes of adenoviral preparations of PTH1R-WT and PTH1R-GC that corresponded to equal surface expression of the receptors, followed by analysis of PTH signaling.

Construction of the pHook-C-Tail Dominant-Negative Constructs
The pHook-1 vector is a discontinued product from Invitrogen that expresses a protein that contains the Ig {kappa}-chain signal peptide, hemagglutinin- and Myc-epitopes, and a single chain antibody linked to the platelet-derived growth factor receptor single-pass transmembrane domain. Using PCR, this cDNA was amplified and cloned into the HindIII and BamHI sites of pcDNA3.1 without a stop codon. The PTH1R C-tail between amino acids 463 and 581 (FC10) and between amino acids 483 and 581 (N20C10) were cloned in to the pcDNA3.1-pHook construct in-frame using the BamHI and XhoI sites.


   ACKNOWLEDGMENTS
 
The authors acknowledge Drs. Henry Kronenberg and John T. Potts for their helpful discussions.


   FOOTNOTES
 
This work was supported by a K01 Grant (K01 DK59900-01) from the National Institute of Diabetes and Digestive and Kidney Diseases (to M.J.M.), Grant GM60286 from the National Institutes of Health (to A.V.S.) and Predoctoral Training Grant HLT3207949 (to T.M.B.).

First Published Online August 11, 2005

Abbreviations: AC, Adenylyl cyclase; GC, GHRHR/PTH1R chimera; GHRHR, GHRH receptor; GPCR, G protein-coupled receptor; GRK, G protein receptor kinase; GTP{gamma}S, guanosine 5'[{gamma}-thio]triphosphate; HEK, human embryonic kidney; HP, double point mutation consisting of W477H and F483P; PLC, phospholipase Cß; PTH1R , PTH-related protein; WW, a double point mutation replacing both conserved tryptophans with alanine.

Received for publication April 25, 2005. Accepted for publication August 4, 2005.


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 DISCUSSION
 MATERIALS AND METHODS
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