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Endocrine Unit, VA Medical Center Departments of Medicine and Physiology, University of California San Francisco, California 94121
Address all correspondence and requests for reprints to: Robert A. Nissenson, Endocrine Unit, VA Medical Center, Departments of Medicine and Physiology, University of California, San Francisco, California 94121.
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Introduction |
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The PTHrP gene was found to be expressed in a wide variety of tissues, but circulating levels of PTHrP were undetectable (or extremely low) in normal individuals, suggesting that PTHrP might serve paracrine or autocrine functions. Subsequent support for this notion derived from the observation that PTHrP is often elaborated by epithelial cells that overlay PTH receptor-expressing mesenchymal cells. Karaplis et al. (6) used molecular genetic technology to generate mice heterozygous for a null PTHrP allele. Such mice were phenotypically normal. However, progeny homozygous for the null allele died shortly after birth, and displayed gross abnormalities in endochondral bone development. In particular, they showed premature ossification in the developing skeleton that manifests as a form of short-legged dwarfism. Histological examination of the growth plate of PTHrP -/- mice revealed markedly reduced numbers of resting and proliferating chondrocytes, suggesting an acceleration in the chondrogenic program. Lanske et al. (7) generated fetal mice with homozygous null mutations in the PTH/PTHrP receptor (PTHR1) gene and found a similar skeletal phenotype-irregular growth plates with reduced numbers of proliferating chondrocytes and premature mineralization.
Three recent papers, one of which appears in this issue of Endocrinology, demonstrate that the PTH/PTHrP receptor also plays a central role in the development of endochondral bone in humans. These papers address the molecular pathogenesis of a rare familial disorder first described in 1985: Blomstrand lethal chondrodysplasia (8). Affected infants display short-limbed dwarfism with markedly accelerated skeletal maturation, increased bone density, and reduced numbers of resting and proliferating chondrocytes in the growth plates of the long bones. Most of the reported cases were born to phenotypically normal, consanguineous parents, suggesting autosomal recessive inheritance. Karaplis et al. (9) and Zhang et al. (10) obtained genomic DNA from one such affected infant, and both groups found a single exonic missense mutation resulting in the conversion of Pro to Leu at position 132 in the N-terminal extracellular domain of the PTH/PTHrP receptor. Only the abnormal allele was detected, indicating the homozygous nature of the mutation. When this mutation was introduced into PTH/PTHrP receptor cDNA and transfected into recipient COS-1 cells, no functional activity was obtained with respect to PTHrP binding or stimulation of cAMP accumulation (9). Expression of this mutant receptor in COS-7 cells resulted in detectable but markedly reduced PTHrP binding and cAMP responses when compared with the wild-type PTH/PTHrP receptor (10). Jobert et al. (11) carried out a similar analysis of a Blomstrand infant from nonconsanguineous parents. The skeletal phenotype in this stillborn infant was similar to that described above. Sequence analysis of genomic DNA demonstrated the presence of one PTH/PTHrP allele with normal exonic sequences, and one allele with a single G to A mutation in exon M5. This mutation creates a new splice acceptor site, resulting in a splice variant encoding a PTH/PTHrP receptor with an 11 amino acid deletion in the fifth transmembrane spanning region. For reasons that are unclear, chondrocytes derived from this infant expressed the abnormal, but not the normal, PTH/PTHrP receptor allele. These chondrocytes failed to respond to PTH with an increase in cAMP production, and the mutant receptor was completely nonfunctional when transiently expressed in COS cells.
What accounts for the loss of functional activity displayed by these mutant PTH/PTHrP receptors? The PTH/PTHrP receptor is a member of the class II subfamily of G protein-coupled receptors (GPCRs). This subfamily includes GPCRs for a number of important peptide ligands in endocrine regulation including secretin, VIP, GHRH, glucagon, GLP-1, and others. Like other members of the GPCR superfamily, the PTH/PTHrP receptor is a single chain protein with seven membrane spanning segments. Existing evidence indicates that activation of the PTH/PTHrP receptor involves cooperative interactions of the ligand with the large N-terminal extracellular domain of the receptor, as well as the carboxy-core domain. Important agonist binding elements in the latter region include the third extracellular loop of the receptor and possibly also the transmembrane spanning segments. According to models developed for the activation of class I GPCRs, agonist binding alters the relative orientation of the receptor’s membrane spanning helices, leading to a conformational change in the cytoplasmic loops permitting activating of the cognate G protein(s). In the case of the PTH/PTHrP receptor (and most other members of the class II subfamily of GPCRs), this would result in activation of adenylyl cyclase (via Gs) and PI-specific phospholipase C (via Gq/G11).
In principle, there are several reasons why mutations in the PTH/PTHrP receptor could produce a loss of function. Mutations could prevent plasma membrane expression of the receptor, either due to protein misfolding and degradation early in the biosynthetic pathway, or because of defects in receptor targeting or lack of stability in the membrane. Such defects have been reported for PTH/PTHrP receptors lacking a C-terminal cytoplasmic tail (12). However, in the study of Karaplis et al. (9), immunohistochemistry of COS cells expressing PTH/PTHrP receptors with the Pro132Leu mutation revealed apparently normal plasma membrane receptor expression. Expression of the mutant receptor was also detected in epiphyseal cartilage from the affected infant. Zhang et al. (10) likewise demonstrated that a fluorescently labeled form of the Pro132Leu receptor mutant was well expressed at the plasma membrane in COS cells. The deletion mutant in the study of Jobert et al. (11) was also found be well expressed on the surface of COS cells. While these studies do not rule out quantitative defects in cell surface expression, they suggest that the mutant PTH/PTHrP receptors were present but were dysfunctional. Pro-132 could represent an important site of contact between the receptor and PTHrP (and PTH). However, this residue does not fall within two domains of the N-terminal region of the PTH/PTHrP receptor that have recently been identified as contact sites for agonists in cross-linking studies (13, 14). It seems most likely that Pro-132 plays an essential role in maintaining the conformation of the N-terminal extracellular domain that is required for high affinity agonist binding and the initiation of signal transduction. Because Pro-132 is a highly conserved residue among class II GPCRs, it will be of considerable interest to determine whether it plays a comparable role in other members of the receptor subfamily. In the case of the mutation described by Jobert et al. (11), it is possible that internal deletion of 11 amino acids in the fifth transmembrane domain renders this region unable to span the plasma membrane. This would grossly alter the conformation of the protein and could even result in important determinants of ligand binding (e.g. the third extracellular loop) facing the cytosolic rather than the extracellular milieu. Even if the deletion allows retention of the basic seven transmembrane domain structure of the receptor, important determinants of ligand binding (15) and signal transduction (16) lie adjacent to the deleted sequence, and the function of these would almost certainly be adversely affected.
Hyperactivation of the PTH/PTHrP receptor in vivo has skeletal consequences that are in many respects the opposite of those seen with loss-of-function receptor mutations. This is apparent from studies in which PTHrP overexpression was targeted to chondrocytes by means of the type II collagen promoter in transgenic mice (17). Severe skeletal abnormalities resulted, characterized by a delay in chondrocyte differentiation and a failure of normal mineralization. Jansen’s metaphyseal chondrodysplasia is a rare dominantly inherited disorder characterized by short-limbed dwarfism due to severe growth plate abnormalities indicative of delayed chondrocyte maturation, hypercalcemia, hypophosphatemia, and increased bone resorption with normal or low levels of circulating PTH and PTHrP. Studies by Schipani and co-workers (18, 19) have demonstrated the presence of heterozygous PTH/PTHrP receptor gene mutations in patients with this disorder. Two point mutations have thus far been described, which encode the conversion of amino acids in putative transmembrane domains of the receptor. Strikingly, expression in COS cells of cDNAs encoding these mutated receptors resulted in increased activity of adenylyl cyclase in the absence of added agonists. This constitutive activity appeared to be limited to the adenylyl cyclase/cAMP signaling pathway because neither mutated form of the receptor produced elevated basal levels of inositol phosphates, the products of phospholipase C activation. The implications of these results are 2-fold. First, constitutive activity of the PTH/PTHrP receptor appears to underlie both the skeletal and the mineral homeostatic defects seen in Jansen’s patients. Secondly, while the PTH/PTHrP receptor is capable of activating both the adenylyl cyclase and phospholipase C signaling pathways, the former pathway is apparently sufficient to induce classical mineral homeostatic responses and skeletal growth plate responses to receptor activation. More definitive support for this conclusion will require demonstration that Jansen’s mutations fail to produce constitutive activation of phospholipase C not only in COS cells but also in physiologically relevant target cells of PTH and PTHrP.
Why do the PTH/PTHrP receptor mutations present in Jansen’s chondrodysplasia result in constitutive receptor activation? Current evidence suggests that, in the absence of agonists, GPCRs are constrained to in inactive conformation due to a series of interactions between amino acids in the transmembrane domains. Agonist binding favors an altered receptor conformation in which these constraining interactions are disrupted, allowing the receptor to activate its cognate G protein(s). One hypothesis is that the residues that are mutated in Jansen’s patients lose the ability to participate in such constraining interactions, allowing a degree of activation in the absence of agonist. One of the mutations is a Thr to Pro conversion at position 410, toward the cytoplasmic end of the sixth transmembrane domain. Strikingly, studies on the activation of rhodopsin, a class I GPCR, indicate that the cytoplasmic ends of transmembrane domains 3 and 6 are in close association with one another in the inactive conformation, and these must become spatially separated in order for activation to occur in response to light (20). Conceivably, Thr-410 participates directly or indirectly in maintaining association of transmembrane domains 3 and 6 in the PTH/PTHrP receptor, and mutation to Pro is sufficient to disrupt this association leading to receptor activation. Indeed, any of a large number of amino acid substitutions at this position produce constitutive activity (21), indicating that Thr-410 plays a very specific role in maintaining the receptor in an inactive state. The other mutation identified in Jansen’s chondrodysplasia is a conversion of His to Arg at position 223 at the cytoplasmic end of the second transmembrane domain. In transfection studies, substitution of Arg or Lys, but not other amino acids at this position resulted in constitutive activity (21), suggesting that a positive charge at this site is required to overcome the inactive receptor conformation in the absence of agonist. It is tempting to speculate that, in the wild-type PTH/PTHrP receptor, agonist binding facilitates the reversible protonation of His-223, resulting in reversible activation and signaling. However, agonist-stimulated activation of adenylyl cyclase does not require a positively charged amino acid at position 223 (21). It seems that the mechanism of receptor activation associated with the Arg-223 mutation differs from that used in response to agonists. Both His-223 and Thr-410 are highly conserved across members of the class II GPCR subfamily, suggesting that their functions are also conserved. Further work is needed to define more precisely the structural roles of Thr-410 and His-223 in regulating the function of the PTH/PTHrP receptor as well as other members of this GPCR subfamily.
These recent studies identifying PTH/PTHrP receptor mutations in human chondrodysplasia provide new insights and raise new questions concerning the biology of endochondral bone development and the molecular mechanisms underlying the ligand binding and signal transduction functions of the PTH/PTHrP receptor. Answers to these questions promise to provide opportunities for the development of new therapeutic approaches for the treatment of a variety of bone and mineral disorders.
Received September 21, 1998.
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