[Frontiers in Bioscience 3, d769-780, August 1, 1998]

Table of Conents
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Wayne B. Bowler1, James A. Gallagher and Graeme Bilbe2

1 Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, University of Liverpool, England, L69 3GE 2 Novartis Pharma A.G., Postfach, K681.4.43, CH-4002 Basel, Switzerland

Received 1/16/98 Accepted 2/17/98


6.1 Calcitonin receptor

The calcitonin receptor (CR) as described above is most closely related to the PTH and secretin receptors that form a distinct subfamily within the G-protein coupled superfamily. There are currently two well characterized CR subtypes that occur through alternative splicing of the CR gene, resulting in the addition or deletion of 16 aa in the first intracellular loop, between the first and second predicted transmembrane helices (56). Although CR display some tissue specificity, osteoclasts express both the insert positive and negative isoforms, however, Northern analysis studies suggest that the insert negative receptor is the predominant isoform (25, 32, 50).

The CR is coupled to both Gq and Gs, initiating pathways that activate both phospholipase C and adenylyl cyclase (100). As such, the peptide hormone calcitonin produced by the parafollicular cells of the thyroid, stimulates cAMP accumulation and induces intracellular calcium elevations in osteoclasts. Recent studies indicate that whilst the binding parameters for both insert negative and positive CRs are indistinguishable, there are striking differences in the ability of these two receptors to couple to second messenger pathways. Insert negative CR alone will mediate rises in intracellular calcium, whilst the ED50 for stimulation of adenylyl cyclase is 100 fold higher for the insert positive CR.

The main function of calcitonin is to negatively regulate calcium homeostasis. As such activation of CR expressed by osteoclasts results in an inhibition of resorption (reviewed in, 87). The mechanism of this inhibition involves both the arrest of osteoclast motility and a shape retraction, termed the Q and R effect respectively. Furthermore, the disruption of F actin ring structures formed at the clear zone of the resorbing osteoclast appears to be a primary mechanism of calcitonin-induced resorption inhibition. While intracellular calcium elevation appears to modulate the R effect (2), F actin disruption occurs following PKA activation (90). It is therefore probable that both the insert positive and negative CRs mediate the osteoclastic effects of calcitonin.

Multiple levels of regulation exists to facilitate CR desensitization. These include receptor-ligand internalization (specific to the insert positive subtype), uncoupling from signaling pathways, down regulation of receptor mRNA expression and loss of receptor binding from the cell surface (reviewed in (20). Specific C-terminal phosphorylation by both PKA, PKC and a non-second messenger dependent receptor kinase (GRK) have recently been reported for both the insert negative and positive CRs, suggesting a role for phosphorylation in the desensitization processes outlined above.

6.2. P2-receptors

It is becoming increasingly apparent that osteoclasts represent targets for extracellular nucleotide activation through G-protein-coupled P2Y receptors. The studies of Yu and Ferrier demonstrated an ATP-induced increase in [Ca2+]i in isolated rabbit osteoclasts, an event attributed to the activation of two pathways; G-protein-mediated release of internal Ca2+ stores, and the opening of Ca2+ channels in the cell membrane facilitating Ca2+ influx (98, 99). However, the nature of the P2Y receptor responsible for this ATP-induced intracellular calcium elevation remains to be elucidated. Recently, Bowler et al, 1998, described the in situ localization of G-protein-coupled P2Y2 receptor transcripts in osteoclasts derived from a human giant cell tumor, however ATP and UTP, agonists at this receptor, were both ineffective at elevating intracellular calcium in isolated giant cells (9). The reasons for the discrepancy in ATP-induced signaling events described in the above studies are unclear, but suggest that in osteoclasts P2Y2 receptors are not coupled through Gq to Ca2+ mobilization, as found in osteoblasts. The coupling of P2Y receptors to signaling mechanisms distinct from PtdIns (4,5)P2 hydrolysis has been reported in other cell types, and include adenylate cyclase, phospholipase D and the mitogen-activated protein kinase cascade (for review see, 5). Further studies are required to elucidate P2Y receptor expression by osteoclasts and to determine receptor/G protein and effector pathway interaction.

Similar uncertainty exists as to the functional role of G-protein-coupled P2Y-receptors expressed by osteoclasts. ATP initiates mechanisms that result in either inhibition or stimulation of bone resorption, depending upon agonist concentration (9, 58). The studies of Bowler et al, 1997, describing ATP, but not UTP-enhanced bone resorption by an enriched population of human giant cells derived from osteoclastoma, would suggest the involvement of an osteoclastic receptor other than the P2Y2 type (9). Indeed, there is mounting evidence that osteoclasts express ionotropic members of the P2-receptor family, including the ATP-responsive P2X4 receptor, although in the absence of receptor-specific antagonists it is difficult to attribute functional effects to activation of particular receptor types.

6.3. Calcium sensing receptor

When exposed to high extracellular Ca2+ concentrations, osteoclasts respond by rapidly elevating intracellular calcium (101). The physiological effects of this event on the osteoclast are profound; dramatic cell retraction followed by an extreme inhibition of resorption (14, 55). As described above a mechanism for sensing extracellular calcium ion concentration involving a G-protein-coupled receptor, the CaR, has been described in the cells of parathyroid and kidney (30). Less clearly defined is the receptor(s) expressed by osteoclasts that sense fluxes in extracellular ion concentration. It is clear from functional binding studies that these receptors are distinct from the molecularly characterized CaR (10), and the putative CaR expressed by osteoblasts (68). Indeed, recent evidence suggests that the sensing mechanism in osteoclasts involves a ryanodine-like calcium release channel (RyR), normally localized in microsomal membranes (102). However, it is unclear if the RyR molecule is physically coupled to and activated by a putative G-protein CaR or alternatively if RyR itself can bind and gate divalent cations. The ability of osteoclasts to sense and functionally respond to rising calcium concentration in their extracellular environment, provides a negative feedback limiting further osteoclast activity, and an intriguing possibility would be the involvement of a molecularly distinct G-protein-coupled receptor.