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Endocrinology Vol. 147, No. 10 4674-4679
Copyright © 2006 by The Endocrine Society

Impact of Impaired Receptor Internalization on Calcium Homeostasis in Knock-In Mice Expressing a Phosphorylation-Deficient Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor

George S. Bounoutas, Hesham Tawfeek, Leopold F. Fröhlich, Ung-il Chung and Abdul B. Abou-Samra

Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Abdul B Abou-Samra, M.D., Ph.D., Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Internalization of G protein-coupled receptors (GPCRs) and desensitization of the hormonal responses are well characterized in vitro for several hormonal systems. The physiological role of internalization for a GPCR receptor involved in homeostatic functions has not been established, although it has been assumed based on in vitro data. We have previously shown that phosphorylation of the PTH/PTHrP receptor is required for its internalization and for the desensitization of the responsiveness to PTH and PTHrP in vitro; the internalization and desensitization response is impaired in a PTH/PTHrP receptor mutant bearing serine to alanine mutations in the phosphate acceptor sites. To understand the physiological role of receptor internalization on calcium homeostasis, we have knocked-in the internalization-impaired PTH/PTHrP receptor mutant using homologous recombination technology. The genetically modified animals exhibited calcium levels no different from control animals, but PTH levels were one third of those in control animals indicating that homeostasis could be maintained only by 3-fold suppression of PTH secretion. We also analyzed the calcemic response to PTH in vivo. Here we show that mice expressing the internalization-impaired PTH/PTHrP receptor mutant have dramatically exaggerated cAMP and calcemic responses to sc PTH administration when compared with control animals given the same dose. These data show for the first time the role of G protein receptor phosphorylation and internalization per se in the regulatory function of an endocrine system controlled by a GPCR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH REGULATES MINERAL ion homeostasis, and PTHrP has an important role in bone development, growth plate organization, and bone cell differentiation. PTH and PTHrP activate a common receptor, the PTH/PTHrP receptor (1, 2) (or PTH1R), expressed in kidney, bone, growth plate cartilage, and several other tissues and cause a transient increase in the intracellular concentrations of the second messengers, cAMP, free calcium, inositol trisphosphate, and diacylglycerol (2, 3).

Internalization and desensitization of G protein-coupled receptors (GPCRs) involve several intracellular molecules (4). Agonist-activated GPCRs are recognized and phosphorylated by specific kinases known as the G protein-coupled receptor kinases (GRKs) (5). GPCRs phosphorylation increases their affinity for ß-arrestins; a group of intracellular proteins that bind phosphorylated GPCRs and clathrin (6). Binding of ß-arrestin to the phosphorylated GPCR results in uncoupling the receptor from the G protein and targets the ligand-receptor complexes to the internalization pathway (4). Internalization results in dissociation of the agonist from the receptor, dephosphorylation of the receptor, and recycling of the receptor back to the cell membrane (recovery) (4), degradation of the receptor, and/or recruitment of new signaling molecules such as MAPKs through protein-protein interactions (7).

Down-regulation of the PTH/PTHrP receptor and desensitization of the cellular responsiveness to PTH have been extensively studied in renal (8, 9, 10, 11, 12) and osteoblastic (13, 14, 15) cells in vitro. Decreased PTH responsiveness has also been reported in vivo using animal models of increased PTH levels and in patients with chronic renal failure (16, 17, 18). Using confocal microscopy and green fluorescent protein-tagged recombinant PTH1R stably expressed in LLCPK-1 cells, we have shown that PTH1R undergoes rapid agonist-dependent internalization within few minutes after PTH challenge (19, 20, 21). Stable expression of PTH1R with a and green fluorescent protein-tagged ß-arrestin revealed a rapid effect of PTH on the redistribution of ß-arrestin between the cytosol and the membrane (22).

Agonist activation of the PTH1R causes phosphorylation of the receptor on serine residues on its carboxyl-terminal tail (positions 489, 491, 492, 493, 495, 501, and 504) (20, 23). Mutation of the seven serine phosphorylation sites to alanine residues resulted in a phosphorylation-deficient PTH1R (pdPTH1R) (20) that does not internalize normally after PTH challenge (20, 24) and that responds to PTH with a sustained increase in intracellular cAMP accumulation (20). The coupling of decreased internalization and sustained elevation of intracellular cAMP indicates the role of receptor phosphorylation in limiting the duration of action of the activated receptor. The physiological relevance of receptor internalization to the endocrine actions of PTH on calcium ion homeostasis and the paracrine actions of PTHrP on bone development, maturation, and turnover have not been previously analyzable. Therefore, to understand the physiological role of the phosphorylation sites, we have developed a mouse model in which the pdPTH1R was knocked-in into the PTH1R locus. We have begun our study of the physiological role of receptor internalization by examining the phenotype of the pdPTH1R mouse and by measuring the serum cAMP and calcium responses to a challenge infusion of PTH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Development of the pdPTH1R mouse
The murine PTH1R gene was cloned from a 129SvJ mouse strain genomic library. A targeting vector was constructed in pcDNA1 that contains an XhoI-EcoRI genomic fragment (fragment 8A, ~4.5 kb) bearing the seven serine to alanine mutations, a neo-tk cassette flanked by LoxP sites from the pFLOX vector, and an EcoRI fragment from the 3' noncoding region of the PTH1R gene (Fragment 8B, ~5.5 kb) (Fig 1AGo). The vector was linearized with XhoI, purified on agaorse gel, and electroporated into embryonic stem (ES) cells (50 µg DNA for 8 x 106 cells in 0.9 ml PBS), which were grown in presence of G418 (300 µg/ml), ES colonies were then screened by Southern blot after KpnI digestion using an external probe (Fig 1BGo). After transient transfection with cre recombinase, ES cell colonies selected in the presence of Gancyclovir (6 µM) were screened for the excision of the neo tk cassette using PCR; the floxed allele is ~450 bp, whereas the wild-type (WT) allele is ~150 bp. Two ES cell colonies were used to generate the knock-in mice. Tail DNA was screened with Southern blot after XbaI digestion (Fig. 1Go, D and E). WT blastocysts were injected with the ES cell lines and chimeric mice were developed, which transmitted the knock-in gene in their germ lines.


Figure 1
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  		FIG. 1. A, Construction of the knock-in vector. The HindIII-EcoRI fragment, encoding the PTH1R carboxy-terminal tail was subcloned in pcDNA1 and mutagenesis was performed to replace the seven serine phosphorylation sites by alanine residues. Mutations were confirmed by sequencing. The 8A fragment was then reconstructed. B, Southern blot of KpnI digestion of genomic DNA from an ES cell line showing homologous recombination. The fragment contained within the knock-in vector is shown as a thick line containing the NeoTK cassette flanked by EcoRI (RI) site. The position of the external probe is also shown. C, PCR amplification using primers flanking the residual LoxP site after CRE digestion. The floxed allele is ~450 bp, whereas the WT allele is ~150 bp. D, Map of the floxed region of the PTH1R gene showing (from bottom to top) WT, knock-in (before CRE) and floxed - after CRE alleles. Digestion with XbaI and Southern blot with the probe shown can differentiate between these three alleles. E, Southern blot of XbaI digested DNA from ES cells before CRE (–CRE) and ES cells after CRE (+CRE); and from mouse tails of pops born from het x het mating. Mice nos. 5, 8, and 9 are homozygous, no. 4 is wt; and the others are het.

 
Primary cultures of osteoprogenitor cells
Bone marrow osteoprogenitor cells were prepared from femurs and tibias of 10-wk-old mice as described (25). The femurs and tibias were dissected free of adherent tissues, the metaphyses were removed and the marrow was flushed from the marrow cavities with {alpha}MEM using a 23-gauge needle. Bone marrow stromal cells were then cultured in {alpha}MEM containing 10% FBS, ascorbic acid (50 µg/ml) and ß-glycerophosphate (10 mM); nonadherent cells were removed by replacing the medium on the next day. The cells were grown for 5 d and then were harvested by trypsinization, seeded in six-well plates at a density of 100,000 cells/well, and grown for 5 d. The cells were then rinsed with serum-free medium, incubated with PTH and 3-isobutyl-1-methylxanthine (IBMX) for the indicated time period as indicated in the figure legends.

PTH infusion
Twelve-week-old male mice were anesthetized by sc injection with ketamine HCl/xylazine HCl solution (Sigma, St. Louis, MO) 2 ml/kg body weight. A small incision was made between the scapulae, and an ALZET osmotic minipump (Durect Corp., Cupertino, CA), with an output of 1 µl/hr, containing vehicle (10 mM acetic acid, 2% heat-inactivated mouse serum) or [Nle23,Tyr34]rPTH(1–34)NH2 [rPTH(1–34)] was implanted sc. The incision was closed with staples. Animals were bled retro-orbitally with heparin-coated capillaries (Bayer, East Wapole, MA) 24, 48, and 72 h after implantation. The serum was spun down at 2500 rpm and kept at –20 C until assayed.

Calcium and phosphate assays
Total serum calcium and total serum phosphate assays were performed in duplicate using Calcium (CPC) Liquicolor and Phosphorous Liqui-UV kits (Standbio, Boerne, TX) as per manufacturer’s instructions.

PTH assay
Plasma PTH levels were measured using a ELISA (Immutopics, Inc., San Clemente, CA). All samples were measured in one assay. The minimum detectable concentration is 5 pg/ml; the intraassay variations are less than 5%.

cAMP assay
Samples diluted in 50 mM acetate (pH 5.5), were acetylated with triethylamine/acetic anhydride (2:1, vol:vol) and were assayed using a double antibody RIA as previously described (2).

1,25(OH)2D3 assay
Plasma samples from 10 WT and eight phosphorylation-deficient (PD) mice, aged 4–6 months, were assayed for 1,25(OH)2D3 using a specific RIA kit ({gamma}-B 1,25-dihydroxy vitamin D RIA; Immuno Diagnostic System Inc., Fountain Hills, AZ) following the manufacturer’s recommendation. Briefly, plasma samples (200 µl) are delipidated and 1,25(OH)2D3 extracted by incubation for 3 h with a solid phase monoclonal anti-1,25(OH)2D3. The immunoextracted 1,25(OH)2D3 is then assayed by a radiocompetition assay using 125I-labeled 1,25(OH)2D3 as a tracer and a highly specific sheep anti-1,25(OH)2D3. Cross-reactivity with 25OHD3 is 0.001% and with 24,25(OH)2D3 is < 0.0123%. All samples were performed in one single assay; intraassay variations are 7.8%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Seven serine to alanine mutations, described for the rat PTH1R (19, 20, 21), were performed on the murine PTH1R cDNA and on genomic DNA cloned from a 129SvJ mouse strain genomic library. The properties of the phosphorylation-deficient murine PTH1R was examined in vitro and was shown to replicate the properties described previously for the rat PTH1R (19, 20, 21). The alanine to serine mutated PTH1R sequences were then knocked in the PTH1R locus using homologous recombination technology (Fig. 1Go). Mating of the heterozygous pdPTH1R mice (pd/wt) resulted in the expected percentage of genotype: 25% WT, 50% heterozygous (pd/wt) for the knock-in gene (pdPTH1R/wtPTH1R) and 25% homozygous for the knock-in gene (pdPTH1R/pdPTH1R or PD) (mice nos. 5, 8, and 9; Fig 1EGo).

To avoid phenotype heterogeneity due to a mixed genetic background, the heterozygous pdPTH1R mouse was backcrossed into the C57/B6J background for six generations. Homozygous PD mice were then obtained from cross mating of heterozygous pdPTH1R mice.

The PD mice show normal appearance, weight, growth, and reproduction. Their basal calcium levels were not different from those of WT littermates (Table 1Go). Strikingly, however, PTH levels were lower in the PD mice than in the WT littermates (14 ± 4 pg/ml in the PD and 46 ± 34 pg/ml in WT; P < 0.05). Also, phosphate levels were significantly lower in the PD mice than in WT littermates (6.39 ± 0.33 mg/dl vs. 7.46 ± 0.33 mg/dl, P < 0.01). The levels of 1,25(OH)2D3 in PD mice were not significantly different from those of the WT littermates: 75.6 ± 7.3 pg/ml (n = 8) and 87.2 ± 15.9 pg/ml (n = 10), respectively.


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  		TABLE 1. Serum levels of ionized calcium, total phosphate, PTH, 1,25(OH)2D3 and weight in 8-wk-old PD and WT male mice.

 
Because the pdPTH1R causes sustained increases in intracellular cAMP accumulation in vitro, we examined whether osteoblasts cultured from the PD mice show sustained increase in cAMP accumulation after PTH challenge (Table 2Go). Primary cultured osteoblasts, prepared from WT and PD mice, were challenged with PTH (10 nM) for 10 min, PTH was removed, and the cells were incubated with culture medium for 5, 10, and 20 min (recovery period). IBMX (5 mM) was then added for an additional 10-min period; and intracellular cAMP accumulation was measured. As found in LLCPK-1 cells (19, 20, 21), the primary cultured osteoblasts from the PD mice showed sustained cAMP accumulation after PTH challenge (Table 2Go).


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  		TABLE 2. Sustained stimulation of cAMP accumulation in osteoblast cultured from PD mice

 
We then examined whether the PD mice show sustained cAMP response to PTH in vivo. Male PD mice and their WT littermates were injected sc with rPTH(1–34) (20 nmol/kg); serum cAMP levels were measured in blood samples collected from the retro-orbital venous sinus. PTH injection caused a transient increase in cAMP accumulation in the WT mice, peaking at 10 min. The PD mice showed a significantly higher serum cAMP levels at all times after the injection and the peak concentration occurred at 15–20 min (Fig 2Go).


Figure 2
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  		FIG. 2. Effects of rPTH(1–34) administration on serum cAMP concentrations in male PD mice. Eight-week-old PD and WT male littermate mice were injected sc with rPTH(1–34) (20 nmol/kg body weight). Retro-orbital venous blood samples were collected at time 0, 5, 10, 15, and 20 min. Serum cAMP concentrations were measured by specific RIA. Vehicle-treated mice show no change in serum cAMP levels. The data are means ± SD calculated from six mice in each group. {star}, P < 0.05 vs. WT.

 
To examine the calcium response to PTH, we measured plasma calcium concentrations after sc infusion of rPTH(1–34) for 3 d. We first determined the effective PTH doses using WT mice (Table 3Go) and found the minimal infusion dose that caused mild hypercalcemia (30 µg/kg·24 h) and then tested this dose in the PD mice to determine whether the response would be exaggerated (Fig. 3Go). PTH infusion caused an exaggerated striking increase in serum calcium concentrations in the PD mice when compared with the WT littermates (Fig 3Go); the peak calcium level after 3-d infusion increased from 12.5 ± 0.7 mg/dl in the WT mice to 22.2 ± 0.8 mg/dl in the PD mice, respectively.


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  		TABLE 3. Effects of increasing doses (0, 3, 30, 300, and 3000 µg/kg/24 h) of PTH(1–34) infusion on serum calcium concentrations in WTmale mice

 

Figure 3
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  		FIG. 3. Effects of rPTH(1–34) infusion on serum calcium concentrations in male WT and PD mice. Twelve-week-old WT (left panel) or PD (right panel) male mice were infused continuously with PTH using osmotic pumps filled with vehicle or rPTH(1–34) (30 µg/kg·24 h) according to manufacturer’s recommendations. The pumps were implanted sc between the shoulder blades under anesthesia. Six mice were used for each group. Total calcium concentrations were measured on retro-orbital blood samples collected at time 0, 24, 48, and 72 h of infusion. The data are means ± SD. {star}, P < 0.01 PD vs. WT.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently ß-arrestin 1 and 2 knockout mice (26) and transgenic mice with targeted overexpression of a peptide inhibitor of GRK2 (27) were used for studying the role of internalization and phosphorylation in GPCR actions in vivo. The targeted expression of a GRK inhibitor in osteoblasts (28) enhanced bone remodeling as well as enhanced urinary excretion of the osteoclastic activity marker, deoxoypyridinoline, with an overall anabolic effect. These are useful models; however, their in vivo phenotype reflects interference with multiple receptor systems. Furthermore, any one receptor, like the PTH1R, probably interacts with multiple GRKs and arrestins, so studying the effects of one GRK or one arrestin at a time gives an incomplete understanding of the PTH1R phosphorylation and internalization (29), and effects of arrestins may not be absolutely dependent on receptor phosphorylation. In contrast, the mutation in the pdPTH1R knock-in mouse model only influences PTH1R action.

The normal blood calcium in the PD mice presumably reflects a homeostatic adjustment leading to a fall of PTH levels to one third of normal to maintain a normal calcium. In that context, the low blood phosphorus levels, despite low PTH levels, suggests increased PTH action to suppress renal proximal tubular phosphate reabsorption. The decreased serum PTH and phosphate concentrations in the PD mice, despite normal calcium and 1,25(OH)2D3 levels; reflects an important physiological function for PTH1R internalization in calcium and phosphate homeostasis.

A single nucleotide C1656T mutation in the PTH1R was recently reported (30) as the genetic cause of Eiken Syndrome, a rare autosomal recessive disorder characterized by multiple epiphyseal dysplasia with extremely retarded ossification of epiphyses, pelvis, hands, and feet and mild growth retardation. The mutation results in a premature termination codon causing deletion of 102 amino acids from the carboxy-terminal tail of the human PTH1R which contains, in addition to the phosphorylated serine residues, several newly recognized functional interactions with NHERF (31, 32), calmudolin (33), calpain (34), and the ß/{gamma}-subunits of the GTP binding proteins (35); these interactions influence the signaling properties of PTH1R and may alter its intracellular degradation. In contrast, the pdPTH1R animal model only examines the role of PTH1R phosphorylation. Because the skeleton of the pdPTH1R mouse does not show gross abnormalities, the skeletal phenotype of the human disease most likely reflects loss of interaction of the other important proteins rather than being a surrogate for the loss of PTH1R phosphorylation.

The finding that the PD mice have decreased PTH and phosphate levels and an exaggerated cAMP and calcium response to PTH administration provides an interesting animal model for an internalization-impaired PTH1R. The model should prove useful in future studies we plan to understand the role of PTH1R internalization in calcium and phosphate homeostasis in animals on different vitamin D and mineral ion dietary supplements. The model will be also very useful to understand the role of PTH1R internalization in the pharmacological response to intermittent vs. continuous PTH administration on bone.


   Acknowledgments
 
We thank Dr. John T. Potts, Jr., for reviewing the manuscript and helpful comments and suggestions.


   Footnotes
 
This work is supported by the National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (Grants RO1 DK062285 and PO1 DK45485).

Disclosure Statement: the authors have nothing to disclose.

First Published Online July 13, 2006

Abbreviations: ES, Embryonic stem; GPCRs, G protein-coupled receptors; GRK, G protein-coupled receptor kinase; IBMX, 3-isobutyl-1-methylxanthine; PD, phosphorylation deficient; pdPTH1R, phosphorylation-deficient PTH1R; PTH1R, PTH/PTHrP receptor; WT, wild type.

Received March 8, 2006.

Accepted for publication July 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals