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. 2008 Jun 24;295(3):E665–E671. doi: 10.1152/ajpendo.00036.2008

Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization

Susanne U Miedlich 1, Abdul B Abou-Samra 2
PMCID: PMC2536737  PMID: 18577695

Abstract

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to Gq/11 proteins upon PTH stimulation predominantly caused by elimination of Ser491/492/493, Ser501, or Ser504. Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to Gq/11 proteins at least partially by direct binding to Gq/11 proteins.

Keywords: G protein-coupled receptor kinases, phosphorylation

the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP (14, 29). Activation of this receptor by PTH plays an important role in calcium and phosphate homeostasis through actions in bone, kidney, and intestine (the latter via production of 1,25-dihydroxyvitamin D). Accordingly, the receptor is most abundantly expressed in osteoblasts and renal tubular cells (14, 29). The PTH1R is also expressed in growth plate chondrocytes, where it controls the normal maturation and organization of these cells (predominantly mediating the autocrine/paracrine actions of PTHrP) (for review see Ref. 23). The crucial physiological importance of PTH1R signaling is emphasized by the dramatic phenotypes of PTH-, PTHrP-, and PTH1R-knockout mice (2, 21, 25, 28).

The PTH1R couples to heterotrimeric Gs, Gi, Gq/11, and G12/13 proteins, which subsequently regulate intracellular effectors such as adenylate cyclase, phospholipases, and mitogen-activated protein kinases (18, 19, 35, 37).

Agonist-dependent activation of G protein-coupled receptors (GPCRs) is generally followed by initiation of a number of regulatory mechanisms leading to rapid signal attenuation, termed functional desensitization. Receptor phosphorylation by GPCR kinases (GRKs) and/or second messenger-dependent protein kinases (PKA or PKC) is regarded as an early step of functional desensitization. Other means of functional desensitization include rapid turnover of second messengers, β-arrestin binding, and receptor internalization.

Upon stimulation, the rat PTH1R becomes phosphorylated at seven serine residues within its COOH-terminal tail (34, 39). Similar findings were reported for the opossum PTH1R; however, only six serine residues were identified (27). Elimination of all seven phosphorylation sites, by serine-to-alanine mutations, results in enhanced and prolonged cAMP formation, as well as impaired internalization of the receptor when stably expressed in LLC-PK1 cells (39).

A mouse model in which a phosphorylation-deficient (pd) receptor was knocked into the locus of the PTH1R (pd PTH1R mouse) reiterates the above-described findings in vivo: subcutaneous injections of PTH led to enhanced cAMP secretion in the pd PTH1R mice compared with wild-type (wt) mice. Continuous infusions of PTH resulted in severe, progressive hypercalcemia in the pd PTH1R, but not wt, mice, which supports the hypothesis that receptor phosphorylation of the PTH1R negatively regulates PTH signaling (4).

We investigated whether activation of phospholipase C (PLC) by the pd PTH1R is altered and whether one or more substitutions of the serine residues identified as PTH-dependent phosphorylation sites of the PTH1R could reproduce the signaling abnormalities observed downstream of the pd PTH1R.

MATERIALS AND METHODS

Materials and plasmids.

[Nle8,21,Tyr34]rPTH(1-34)NH2 [PTH, or PTH(1-34)] was kindly provided by A. Khatri (Endocrine Unit, Massachusetts General Hospital, Boston, MA). Chemicals were obtained from Sigma-Aldrich (St. Louis, MO). The rat wt PTH1R cDNA cloned into pcDNA1, R15B (1), was used for single-strand plasmid preparation. Single or multiple alanine mutations were introduced by site-directed mutagenesis (24) at positions 489, 491, 492, 493, 495, 501, and 504 within the COOH-terminal tail of the PTH1R to produce the following receptor mutants: S489A, S501A and S504A, S491/492/S493A, S492/493/495A, S489/491/492/493/495A, and S489/491/492/493/495/501/504A (pd PTH1R). These residues were phosphorylated upon stimulation with PTH, as shown previously (39). The mutations were verified by sequencing. All mutants were examined for expression, and all have an expression level similar to or higher than that of the wt receptors. Plasmids of bovine GRK2, GRK3, GRK5, and GRK6 were kindly provided by Dr. J. L. Benovic (Thomas Jefferson University, Philadelphia, PA), and plasmids containing D110A-GRK2 or K220R-GRK2 were a gift from Dr. S. S. Ferguson (Robarts Research Institute, London, ON, Canada).

Cell culture and transfections.

COS-7 cells were cultured in DMEM (Mediatech, Manassas, VA) supplemented with 10% FBS, 100 U/ml penicillin, and 1 μg/ml streptomycin at 37°C and 5% CO2. Cells were transiently transfected with different receptor constructs (2 μg total DNA/well of 6-well plate) using diethylaminoethyl-dextran (Sigma-Aldrich) according to the manufacturer's instructions. In all cotransfection experiments, the total amount of plasmid DNA was kept constant (2 μg/well) by addition of empty vector DNA or the second plasmid of interest at the desired ratio.

Cell surface receptor quantification.

Quantification of cell surface receptors was performed as previously described (39). Briefly, COS-7 cells were plated onto 24-well plates 24 h after transfection and assayed 36–48 h later. After two rinses with Hanks' balanced salt solution (Mediatech), cells were incubated with a receptor-specific antiserum (G48; 1:2,000 dilution) (34) for 120 min at 4°C. After a second rinse, a rabbit anti-sheep IgG antiserum (1:500 dilution; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the cells for 90 min at room temperature. Cells were rinsed again before a 125I-labeled donkey anti-rabbit IgG F(ab′)2 fragment (∼250,000 cpm/well; GE Healthcare, Buckinghamshire, UK) was added for another 90 min at room temperature. Cells were then rinsed three times and solubilized with 1 N NaOH, and radioactivity was counted in a gamma counter (model 6/400 Plus, Micromedic Systems, Horsham, PA). Receptor levels are expressed as percentage of total radioactivity bound (after subtraction of background radioactivity measured in vector-transfected COS-7 cells) normalized to basal or wt receptor levels depending on the experiment.

Inositol phosphate assays.

Inositol phosphates were measured as previously described (41). Briefly, 48 h after transfection, the cells were incubated with 2 μCi/ml [myo-3H]inositol (18.6 Ci/mmol, Amersham, Buckinghamshire, UK) in serum- and myo-inositol-free DMEM for 12 h. Subsequently, the cells were stimulated with or without PTH in serum-free DMEM containing 20 mM HEPES (pH 7.4), 0.1% BSA, 100 mg/ml aprotinin, and 1 mM LiCl2. The stimulation was terminated by aspiration of the medium and addition of 3% perchloric acid. Intracellular inositol phosphates were separated by anion-exchange chromatography, and the radioactivity was determined by liquid scintillation counting in a liquid scintillation counter (Beckman Coulter, Fullerton, CA). Inositol phosphate values are expressed as the ratio of radioactivity incorporated into inositol phosphates to total radioactivity incorporated into both inositol phosphates and phosphatidyl inositols.

Data analysis.

Inositol phosphate and receptor expression values calculated as described above were normalized to the control condition within each experimental setting and subsequently used for statistical analysis. All statistical/graphical analyses were done with Excel (Microsoft, Redmond, WA) or PRISM 3.02 (GraphPad, San Diego, CA). ANOVA followed by t-tests was utilized to compare experimental conditions using the different receptor/GRK constructs. Curves were fitted using classic equations for hyperbolic dose responses (PRISM 3.02). P < 0.05 was considered statistically significant.

RESULTS

PTH-dependent activation of the pd PTH1R leads to higher inositol phosphate accumulation.

Upon PTH treatment, ligand binding and cAMP accumulation were not significantly different in COS-7 cells transfected with the wt or pd PTH1R (39) (data not shown).

In COS-7 cells, PTH1R stimulation leads to activation not only of adenylate cyclase but also, although less efficiently, of PLC. Since ligand and receptor conformations differentially affect downstream signaling pathways for the PTH1R (3, 6, 7, 37), we asked whether PLC stimulation was altered in cells expressing the pd PTH1R mutant under basal conditions or upon ligand stimulation. For this purpose, COS-7 cells were transiently transfected with the wt or pd PTH1R and assayed for inositol phosphate formation after PTH treatment. Initial time course experiments using 10−7 M PTH indicated that inositol phosphate accumulation was significantly increased in cells transfected with the pd PTH1R compared with the wt PTH1R, with the differences being more enhanced after 1 h than after 30 min of incubation (Fig. 1A). Therefore, 1-h incubations were chosen for all subsequent experiments. Upon stimulation with increasing concentrations of PTH, maximal inositol phosphate accumulation (Rmax) was increased by about threefold in COS-7 cells transfected with the pd PTH1R compared with cells expressing the wt receptor, whereas the EC50 was virtually indistinguishable: EC50 = 6.37 × 10−9 M and Rmax = 3.24% for the wt PTH1R and EC50 = 9.19 × 10−9 M and Rmax = 8.93% for the pd PTH1R (Fig. 1B). Basal inositol phosphate levels were not different in cells transfected with the wt or pd PTH1R.

Fig. 1.

Fig. 1.

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Inositol phosphate (IP) accumulation mediated by phosphorylation-deficient (pd) parathyroid hormone (PTH)-related peptide receptor (PTH1R) is dramatically increased upon PTH treatment. A: COS-7 cells transiently transfected with wild-type (wt, •) or pd (○) PTH1R were stimulated with 10−7 M PTH, and inositol phosphate accumulation was evaluated after 30 and 60 min of incubation in the presence of 1 mM LiCl2. Values are means ± SE of 3 independent transfections. B: COS-7 cells transiently transfected with wt or pd PTH1R were stimulated with increasing concentrations of PTH in the presence of LiCl2, and inositol phosphate accumulation was determined after 1 h. Values are means ± SE of 3 independent transfections. Curves were fitted using classic equations for hyperbolic dose responses: y = Rmax × x/(EC50 + x), where Rmax is maximal inositol phosphate accumulation. For wt PTH1R, Rmax = 3.24% and EC50 = 6.37 × 10−9 M; for pd PTH1R, Rmax = 8.93% and EC50 = 9.19 × 10−9 M.

Individual mutations of PTH1R phosphorylation sites differentially affect inositol phosphate accumulation upon PTH treatment.

Next, we examined whether any single or combined substitution(s) of the seven serine residues could reproduce the above-described findings. COS-7 cells were transfected with wt PTH1R or pd PTH1R constructs with single or triple amino acid mutations, stimulated with 10−7 M PTH, and assayed for inositol phosphate accumulation.

PTH-dependent inositol phosphate accumulation was significantly increased for the PTH1R mutants S491/492/493A, S501A, and S504A, but not S489A and S492/493/495A, compared with the wt receptor (Fig. 2). This result suggests that preventing phosphorylation of S491/492/493, S501, or S504 is sufficient to enhance coupling of the PTH1R to PLC upon ligand treatment. However, inositol phosphate accumulation in cells transfected with S491/492/493A, S501A, or S504A was still significantly lower than the response observed for the pd PTH1R (P < 0.05), suggesting that the individual mutations do not fully recapitulate the phenotype of the pd PTH1R.

Fig. 2.

Fig. 2.

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Individual mutations of PTH1R phosphorylation sites differentially affect inositol phosphate accumulation upon PTH treatment. COS-7 cells transiently transfected with wt PTH1R or pd PTH1R constructs were stimulated with 10−7 M PTH in the presence of LiCl2, and inositol phosphate accumulation was determined after 1 h. Values are means ± SE of ≥3 independent transfections (normalized to wt PTH1R). *P < 0.05; ***P < 0.001 vs. wt.

Previous studies in LLC-PK1 cells stably expressing wt PTH1Rs have shown that PTH-mediated PLC stimulation is directly dependent on the number of receptors expressed on the cell surface (16). We therefore hypothesized that increased basal receptor expression may be responsible for enhanced coupling to PLC. Basal expression was significantly increased by 20–50% for all the mutant receptors (Fig. 3) and could contribute to the observed enhancement in inositol phosphate formation for the different receptor constructs. To test this hypothesis, we measured inositol phosphate formation as well as receptor expression in the same transfection. For each experimental setting, the inositol phosphate response was normalized to basal receptor levels and subsequently compared with the wt PTH1R. Adjusted inositol formation in response to PTH treatment was significantly increased in cells transfected with the pd PTH1R, S491/492/493A, S501A, or S504A compared with those transfected with the wt PTH1R; these data likely reflect an intrinsic property of the pd receptors (Fig. 4).

Fig. 3.

Fig. 3.

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Expression of pd PTH1R constructs is moderately increased compared with wt receptors. COS-7 cells transiently transfected with wt PTH1R or pd PTH1R constructs were incubated in serum-free DMEM containing 20 mM HEPES (pH 7.4), 0.1% BSA, and 100 mg/ml aprotinin for 1 h at 37°C. After 2 rinses with Hanks' balanced salt solution, cell surface receptors were quantified as described previously (39). Values are means ± SE of ≥3 independent transfections (normalized to wt PTH1R). *P < 0.05; ***P < 0.001 vs. wt. Specific radioactivities (means ± SE from ≥3 independent transfections) for individual receptor mutants were as follows (counts/min): 8,033 ± 1,201 for wt, 9,747 ± 1,234 for pd, 9,924 ± 865 for S489A, 10,144 ± 656 for S491/492/493A, 9,825 ± 481 for S492/493/495A, 11,338 ± 790 for S501A, and 11,339 ± 360 for S504A.

Fig. 4.

Fig. 4.

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Individual mutations of PTH1R phosphorylation sites differentially affect inositol phosphate accumulation upon PTH treatment independent of basal receptor expression. COS-7 cells transiently transfected with wt PTH1R or pd PTH1R constructs were evaluated for basal receptor levels 72 h after transfection as described elsewhere (39). Cells were subsequently stimulated with 10−7 M PTH in the presence of LiCl2, and inositol phosphate accumulation was determined after 1 h. Inositol phosphate responses were then normalized to basal receptor levels and compared with those of wt PTH1R. Values are means ± SE of ≥3 independent transfections. *P < 0.05; **P < 0.01; ***P < 0.001 vs. wt.

Receptor internalization is impaired in COS-7 cells transiently expressing the pd PTH1R.

We previously demonstrated that internalization of the pd PTH1R is significantly impaired in LLC-PK1 cells stably expressing this mutant receptor (39). We therefore studied the internalization properties of the pd PTH1R in this transient expression system. In COS-7 cells transiently transfected with wt or pd PTH1R, receptor internalization reached a maximum 15–30 min after PTH addition and did not significantly change up to 1 h (data not shown). Therefore, we selected 1 h for comparison of inositol phosphate production and cell surface receptor levels. Receptor internalization after PTH treatment was significantly less for the pd PTH1R (all 7 serines substituted by alanines) than for the wt PTH1R after 30 min (data not shown) and 60 min (Fig. 5) of agonist incubation, confirming our previously published observations (39). However, none of the single or triple phosphorylation site mutants showed significantly altered internalization compared with the wt receptor (Fig. 5). We also tested a mutant receptor in which five serine residues were replaced by alanine residues (S489/491/492/493/495A), and again internalization upon PTH stimulation was not significantly different from the wt PTH1R when analyzed under the above-described conditions (Fig. 5). Collectively, our findings suggest that as few as one or two phosphorylated serine residues may be sufficient to mediate agonist-dependent internalization of the PTH1R.

Fig. 5.

Fig. 5.

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Receptor internalization is impaired for pd PTH1R. COS-7 cells transiently transfected with wt PTH1R or pd PTH1R constructs were incubated in serum-free DMEM containing 20 mM HEPES (pH 7.4), 0.1% BSA, and 100 mg/ml aprotinin with or without 10−7 M PTH for 1 h at 37°C. After 2 rinses with Hanks' balanced salt solution, cell surface receptors were quantified as described elsewhere (39). Values are means ± SE of ≥3 independent transfections (normalized for each construct to receptor levels without PTH). *P < 0.05 vs. wt.

GRK overexpression impairs inositol phosphate accumulation by interfering with pd PTH1R Gq/11 protein coupling.

Formation of wt PTH1R-mediated inositol phosphate can be inhibited by GRK2, GRK3, and GRK5 overexpression (11). To obtain more mechanistic insights into how GRKs interfere with PTH-mediated PLC stimulation, we asked whether overexpression of the ubiquitously expressed GRK2, GRK3, GRK5, and GRK6 could interfere with increased inositol phosphate production through the pd PTH1R. We therefore expressed the wt or pd PTH1R together with GRK2, GRK3, GRK5, or GRK6 (4:1 receptor-to-GRK ratio) and measured inositol phosphate formation in response to stimulation with 10−7 M PTH. Overexpression of GRK2, GRK3, GRK5, and GRK6 significantly reduced inositol phosphate production downstream of the wt or pd PTH1R. The magnitude of this effect on PLC stimulation did not differ for the wt or pd PTH1R (Fig. 6, for comparison of the wt and pd PTH1R, see reference line drawn at 50% inhibition of either control condition). This result suggests that all ubiquitously expressed GRKs interfere with PLC stimulation most likely independent of PTH1R phosphorylation and that receptor phosphorylation and functional desensitization by GRKs may be independent events at least with regard to PLC activation. Dicker and co-workers (11) demonstrated that both wt and a catalytically inactive GRK2 similarly inhibited PTH-mediated PLC stimulation through the PTH1R. Overexpression of the COOH terminus of GRK2 (aa 495-689) did not interfere with PTH-dependent PLC stimulation (11). The latter findings suggest that GRK2 may interfere with PLC activation through its relatively conserved NH2 terminus (33). The NH2 terminus of GRKs has been proposed to contain elements for GPCR binding (30). GRK2 and GRK3, via their NH2-terminal domains, also comprise a Gq/11 protein-binding site by which they directly interact with Gq/11 proteins in an AlF4−-dependent manner (5). We therefore reasoned that the inhibitory effect of GRK2 overexpression on inositol phosphate production could be mediated by direct binding of GRK2 to Gq/11 proteins, as demonstrated for other GPCRs (8, 13, 20, 22, 26, 32). To test this hypothesis, we expressed either the wt or pd PTH1R together with wt GRK2 or mutant GRK2 constructs defective in Gq/11 protein binding (D110A) or catalytic activity (K220R) (36). In cells expressing the wt PTH1R, wt and mutant GRK2s (D110A and K220R) significantly inhibited PTH-dependent inositol phosphate production, suggesting that catalytic activity and direct binding of GRK2 to Gq/11 proteins were not necessary for GRK2 inhibition of PLC activation (Fig. 7). However, because of the low coupling efficiency of the wt receptor to Gq/11 protein (2- to 4-fold increase over basal inositol phosphate production with 10−7 M PTH in control condition), it is difficult to draw definitive conclusions from these experiments. Thus we coexpressed the pd PTH1R, which shows enhanced coupling to Gq/11 proteins, as shown earlier, with wt GRK2, K220R-GRK2, or D110A-GRK2. As demonstrated for the wt PTH1R, wt GRK2 and K220R-GRK2 significantly inhibited inositol phosphate formation in cells transfected with the pd PTH1R, suggesting that GRK2 effects on PTH-dependent PLC signaling are independent of receptor phosphorylation, as shown previously (11). However, overexpression of D110A-GRK2 reduced inositol phosphate formation to a significantly lesser extent than wt GRK2 (pd PTH1R + wt GRK2 vs. pd PTH1R + D110A-GRK2, P < 0.05; Fig. 7). These findings imply that inhibition of PTH-dependent inositol phosphate formation by GRK2 involves, in part, a direct interference of the kinase with Gq/11 proteins, at least for the pd PTH1R.

Fig. 6.

Fig. 6.

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G protein receptor-coupled kinases (GRK2, GRK3, GRK5, and GRK6) inhibit inositol phosphate accumulation independent of PTH1R phosphorylation. COS-7 cells transiently transfected with wt or pd PTH1R and pcDNA or GRK2, GRK3, GRK5, and GRK6 were stimulated with 10−7 M PTH in the presence of LiCl2, and inositol phosphate accumulation was determined after 1 h. Values are means ± SE of 3 independent transfections normalized to control (wt + pcDNA). *P < 0.05; **P < 0.01; ***P < 0.001 vs. receptor (wt or pd) + pcDNA.

Fig. 7.

Fig. 7.

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GRK2 inhibits pd PTH1R-mediated inositol phosphate accumulation at least partially through direct binding to Gq/11 proteins. COS-7 cells transiently transfected with either wt or pd PTH1R and pcDNA or wt GRK2 or GRK2 mutants deficient in Gq/11 protein binding (D110A) or catalytic activity (K220R) were stimulated with 10−7 M PTH in the presence of LiCl2, and inositol phosphate accumulation was determined after 1 h. Values are means ± SE of 3 independent transfections normalized to control. *P < 0.05, pd PTH1R + wt GRK2 vs. pd PTH1R + D110A.

DISCUSSION

The present study explored the role of individual PTH1R phosphorylation sites in PTH1R signaling through PLC, agonist-dependent receptor internalization, and regulation by GRKs. We demonstrated by means of a heterologous expression system that PLC activation is dramatically enhanced downstream of the pd PTH1R and that this is not secondary to increased receptor numbers on the cell surface. It is conceivable, however, that impairment of agonist-dependent internalization may contribute to the dramatic enhancement of PLC stimulation of the pd PTH1R. However, internalization of the individual receptor mutants S491/492/493A, S501A, and S504A, which also showed enhanced PLC responses/receptors (Fig. 4), was not significantly different from wt receptors (Fig. 5). The latter findings suggest that increased inositol phosphate production mediated by these mutant PTH1Rs may reflect enhanced and/or prolonged coupling to Gq/11 proteins. Disclosure of the exact molecular mechanisms for such a prolonged and enhanced coupling requires further investigation. The requirement for Gq/11 proteins for the enhanced PLC stimulation by the pd PTH1R was recently established by the demonstration that small interfering RNA inhibition of Gq/11 protein expression in LLC-PK1 cells stably expressing the pd PTH1R dramatically decreased PLC stimulation by the pd PTH1R (38).

Consistent with our conclusions, we have also shown that enhanced PLC activation of the pd PTH1R can only partially be impaired by overexpression of GRK2 mutants that cannot interfere with receptor-Gq/11 coupling. Moreover, all ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6), as well as a catalytically inactive GRK2 mutant (K220R), were able to reduce PLC stimulation downstream of the pd PTH1R, supporting the model that GRKs may interfere with PTH-dependent PLC stimulation in a phosphorylation-independent manner (11). Such concerted interactions of GRKs, G proteins, and GPCRs have been described for the metabotropic glutamate receptor type 1 (mGluR1) (8–10); two basic residues within the second intracellular loop and the COOH terminus of mGluR1 (Lys691 and Lys692) were identified as binding sites for GRK2. For the α2-adrenergic receptor, basic residues (Arg125, Lys320, and Lys358) within the third intracellular loop of mGluR1 have been reported to disrupt receptor-GRK2 interactions (31). Although all these studies point to ionic interactions between GPCRs and GRKs, they also indicate that distinct domains contribute to GPCR family-specific GRK interactions (for review see Ref. 12). It is of interest to note that the COOH-terminal domain of the PTH1R directly proximal to the phosphorylation sites (aa 484-487) consists of four highly basic residues that could potentially act as a binding site for GRKs, thereby interfering with Gq/11 protein coupling. Further studies are necessary to dissect the mechanisms by which GRKs interfere with signaling of family B GPCRs.

In addition to enhanced signaling through Gs and Gq/11 proteins, the pd PTH1R is significantly impaired in agonist-dependent internalization (39) (Fig. 5). Interestingly, this observation is only evident upon mutation of all seven serine residues to alanine residues. In addition, mutation of up to five phosphorylation sites did not negatively affect agonist-dependent internalization, suggesting that phosphorylation of any one or two serine residue(s) within the COOH-terminal tail of the PTH1R may be sufficient to promote agonist-stimulated receptor internalization. On the basis of the results, it is also possible that phosphorylation of Ser501 and Ser504 is necessary to mediate agonist-dependent receptor internalization.

Is there evidence that the distinct characteristics of pd PTH1R signaling contribute to regulation of mineral metabolism in vivo? Young male pd PTH1R mice are hypophosphatemic and hyperphosphaturic, pointing to enhanced and/or prolonged PTH1R actions within renal proximal tubules (4; S. U. Miedlich, unpublished observations). Regulation of renal phosphate handling by PTH is mediated by PKA- and PKC-dependent pathways (40). It is therefore possible that altered phosphate handling in the pd PTH1R mice may at least partly reflect enhanced PTH1R signaling via Gq/11 and/or Gi protein-mediated pathways. Further studies with signaling-selective agonists and mouse models with selective PTH1R signaling (15, 17) are required to address whether and to what extent altered PLC signaling contributes to regulation of phosphate homeostasis by PTH. In fact, preliminary data (17) point to altered phosphate handling under conditions of chronic PTH stimulation (induced by calcium deficiency) in DSEL mice, which are selectively deficient in PLC but not cAMP-dependent signaling of the PTH1R.

In conclusion, our work provides evidence that the pd PTH1R, as well as PTH1R mutants S491/492/493A, S501A, or S504A, confer significantly enhanced coupling to Gq/11 proteins upon PTH stimulation. On the other hand, reportedly impaired internalization of the pd PTH1R was not dependent on a specific phosphorylation site. In addition, we show here that GRK2, GRK3, GRK5, and GRK6 interfere with pd PTH1R signaling to Gq/11 proteins. The latter mechanism could potentially play a role as an adaptive mechanism in pd PTH1R-knockin mice, which outgrow the hypophosphatemic phenotype by 12 wk of age (S. U. Miedlich, unpublished observations).

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-063211 and DK-11794.

Acknowledgments

The authors thank Drs. Hesham A. Tawfeek, John T. Potts, Jr., Marie B. Demay, and Ernestina Schipani for continuous encouragement, critical discussions of the data, and careful review of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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