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S R Goldring

Publications and source records attributed to S R Goldring.

At least 37 records · Page 2Linked to original sources
PubMed1998

Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor.

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.

Animals↗
PubMed1998

Identification of cell types responsible for bone resorption in rheumatoid arthritis and juvenile rheumatoid arthritis.

Focal resorption of bone at the bone-pannus interface is common in rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA) and can result in significant morbidity. However, the specific cellular and hormonal mechanisms involved in this process are not well established. We examined tissue sections from areas of bone erosion in patients with RA and JRA. Multinucleated cells (MNCs) were present in resorption lacunae in areas of calcified cartilage and in subchondral bone immediately adjacent to calcified cartilage, as previously described. mRNA for the calcitonin receptor (CTR) was localized to these MNCs in bone resorption lacunae, a finding that definitively identifies these cells as osteoclasts. These MNCs were also positive for tartrate-resistant acid phosphatase (TRAP) mRNA and TRAP enzymatic activity. Occasional mononuclear cells on the bone surface were also CTR positive. Mononuclear cells and MNCs not on bone surfaces were CTR negative. The restriction of CTR-positive cells to the surface of mineralized tissues suggests that bone and/or calcified cartilage provide signals that are critical for the differentiation of hematopoietic osteoclast precursors to fully differentiated osteoclasts. Some MNCs and mononuclear cells off bone and within invading tissues were TRAP positive. These cells likely represent the precursors of the CTR-TRAP-positive cells on bone. Parathyroid hormone receptor mRNA was present in cells with the phenotypic appearance of osteoblasts, in close proximity to MNCs, and in occasional cells within pannus tissue, but not in the MNCs in bone resorption lacunae. These findings demonstrate that osteoclasts within the rheumatoid lesion do not express parathyroid hormone receptor. In conclusion, the resorbing cells in RA exhibit a definitive osteoclastic phenotype, suggesting that pharmacological agents that inhibit osteoclast recruitment or activity are rational targets for blocking focal bone erosion in patients with RA and JRA.

Acid Phosphatase↗
PubMed1997

Multinucleated cells in pigmented villonodular synovitis and giant cell tumor of tendon sheath express features of osteoclasts.

Pigmented villonodular synovitis (PVNS) and the histologically related lesion giant cell tumor of tendon sheath (GCTTS) are idiopathic, proliferative lesions that can induce osteolysis and formation of bone cysts. These lesions contain two predominant cell types: mononuclear polyhedral cells and multinucleated cells (MNCs). Previous studies demonstrated that the mononuclear cells exhibit phenotypic features consistent with derivation from a monocyte/macrophage lineage. The cell lineage of the MNCs and their relationship to osteoclasts are not known. To characterize the MNCs in these lesions and to establish the relationship of these MNCs to osteoclasts, histological sections from six cases of PVNS and two cases of GCTTS were studied. Mononuclear cells expressed CD14 and HLA-DR, in keeping with their relationship to cells of the monocyte/macrophage lineage. Characterization of the MNCs revealed features associated with an osteoclast phenotype. Seven of the eight specimens contained MNCs that were intensely tartrate-resistant acid phosphatase positive; approximately 5% of the mononuclear cells were tartrate-resistant acid phosphatase positive, and these tended to surround MNCs. MNCs in both lesions reacted strongly with the 23C6 monoclonal antibody that recognizes the alpha V beta 3 integrin (the vitronectin receptor), as did several mononuclear cells surrounding the MNCs. Most MNCs did not express CD14 or HLA-DR. Expression of receptors for calcitonin, a marker for osteoclasts, was detected on MNCs after incubation of sections with 125I-labeled salmon calcitonin and emulsion autoradiography. MNCs in four of six PVNS and two of two GCTTS samples demonstrated specific calcitonin binding. Expression of mRNA for calcitonin receptor was confirmed in all cases by reverse transcriptase polymerase chain reaction. These results demonstrate that MNCs in PVNS and GCTTS express phenotypic features of authentic osteoclasts and suggest that osteoclast-like multinucleated cells can arise in synovial soft tissues remote from bone.

Acid Phosphatase↗
PubMed1997

Design, synthesis and utility of novel benzophenone-containing calcitonin analogs for photoaffinity labeling the calcitonin receptor.

Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.

Amino Acid Sequence↗
PubMed1996

Full activation of chimeric receptors by hybrids between parathyroid hormone and calcitonin. Evidence for a common pattern of ligand-receptor interaction.

Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G protein-coupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wild-type" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.

Animals↗
PubMed1996

Differential effects of different forms of hydroxyapatite and hydroxyapatite/tricalcium phosphate particulates on human monocyte/macrophages in vitro.

A possible complication associated with the use of hydroxyapatite (HA) or HA/tricalcium phosphate (HA/TCP) coating on the surfaces of prosthetic devices used for dental and orthopedic implants is their potential to fragment and thus exist as wear debris. In contrast to the so-called osteoconductive properties of HA or HA/TCP coatings, in particulate form these materials may lead to an adverse pattern of cellular and tissue responses at the bone-implant interface. We have established an in vitro cell culture system to characterize the biologic and biochemical effects of various particulate materials. The present study demonstrates that the HA/TCP particles derived from different sintering temperatures exhibit differential effects on cultured human monocyte/macrophages (M/M). The HA/TCP particles dried at 110 degrees C were the most biologically active, stimulating significant release of interleukin 1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha, and prostaglandin E2 (PGE2), products implicated as important mediators of inflammation in diverse pathologic conditions. Other particles, sintered at either 900 or 1200 degrees C, did not stimulate production of cytokines or PGE2. HA/TCP particles from plasma-spray coatings also failed to release proinflammatory products. These results suggest that the biochemical and crystalline structural properties of particles markedly affects their capacity to modulate M/M function. This in vitro culture system should be useful in characterizing the specific physical and chemical properties of HA or HA/TCP particulates that are responsible for stimulating proinflammatory cell responses.

Biocompatible Materials↗
PubMed1996

Serpentine receptors for parathyroid hormone, calcitonin and extracellular calcium ions.

The cloning of the receptors for PTH, CT and extracellular calcium ions represents a significant advance in the elucidation of the mechanisms through which extracellular calcium ions are regulated. All are members of the superfamily of GPCR, and the inclusion of the Ca2+o-sensing receptor in this superfamily documents that extracellular calcium ions can serve as an extracellular first messenger, in addition to subserving their better known role as a key intracellular second messenger. Furthermore, it has proved possible to identify several human diseases that result from inactivating or activating mutations in the PTH or Ca2+o-sensing receptor. Finally, the availability of these cloned receptors will enable many more studies on structure-function relationships for these receptors as well as clarifying their tissue distribution, regulation and roles in health and disease. It may also be possible to design novel therapeutic agents that permit manipulation of the receptors when their function is abnormal.

Animals↗
PubMed1996

Cytokines and skeletal physiology.

Cytokines are soluble factors that play a critical role in mediating cell to cell interactions within skeletal tissues. These effects are mediated by paracrine, autocrine, and juxtacrine mechanisms. There are also examples in which the cytokines can function in an endocrine fashion. The regulatory functions of the cytokines are performed throughout life, beginning with bone growth and development and continuing in the mature organism, in which bone remodeling is regulated. The cytokines can be grouped into distinct families based on their principal biologic activity and cell target. However, the activities of the cytokines are pleiotropic, and they exhibit considerable overlap and redundancy in their actions. The molecular cloning of the cytokines and their receptors and elucidation of their common structural features have aided in the understanding of the molecular basis for the redundancy and pleiotropy of cytokine effects. Examination of most physiologic processes in which cytokines play an important regulatory role reveals that cytokines rarely exert their biologic activities in isolation. Instead, these soluble factors usually are produced locally in concert with many other cytokines. The interaction of these structurally and functionally distinct factors in a highly ordered temporal and spatial sequence creates a cytokine network that ultimately determines a given tissue's response. Continued investigation into the molecular and biologic mechanisms by which cytokines regulate bone cell function will provide additional insights into normal bone physiology and permit more effective and specific use of these factors in the treatment of skeletal disorders.

Animals↗
PubMed1995

Calcitonin increases cytosolic free calcium concentration via capacitative calcium influx.

The calcitonin receptor has been proposed to function as an extracellular Ca2+ concentration ([Ca2+]o) sensor (Stroop, S. D., Thompson, D. L., Kuestner, R. E., and Moore, E. E. (1993) J. Biol. Chem. 268, 19927-19930). To test this hypothesis we studied the LLC-PK1 renal tubular cells and the PC1 cells, a cell line stably transfected with the cloned porcine calcitonin receptor. [Ca2+]i was measured by fura-2 single cell microfluorometry. Addition to the cells equilibrated in 1.25 mM Ca(2+)-containing media of 1-10 mM extracellular Ca2+ did not result in a significant increase of [Ca2+]i. Treatment with 10(-7) M salmon calcitonin (sCT) elicited a rapid, persistent elevation of [Ca2+]i. Addition of 1-10 mM extracellular Ca2+ in the presence of sCT induced a significant [Ca2+]i elevation, about 10-fold that observed in the absence of the hormone. Ca2+ influx was inhibited by lanthanum. The rise of [Ca2+]i at elevated [Ca2+]o was not due to a Ca2+ sensing mechanism with release of Ca2+ from intracellular stores, since it was prolonged, and was not abolished by prior depletion of Ca2+ stores with 10(-6)M thapsigargin. On the contrary, this agent potentiated Ca2+ influx after addition of 1-10 mM Ca2+ by 13-fold versus control. Prior stimulation of [Ca2+]i with 10(-7) M arginine-vasopressin had similar effects, enhancing the subsequent Ca2+ influx. Enhancement of Ca2+ influx by sCT was confirmed by increased Mn2+ quenching of fura-2 fluorescence. In conclusion, arginine-vasopressin or calcitonin enhance Ca2+ influx in LLC-PK1 cells via a Ca2+ release-activated conductance, probably dependent upon capacitative Ca2+ entry. Thus, these effects are not unique to the calcitonin receptor and argue against the receptor functioning as a [Ca2+]o sensor.

Animals↗
PubMed1995

Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction.

Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system.

Animals↗
PubMed1995

Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin.

We studied the temporal sequence of osteoclast (OC) differentiation from precursor cells in murine marrow cultures. Two markers of the OC phenotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid phosphatase (TRAP), were assessed. Marrow cells from C57BL/6 mice were cultured for 3, 5, 7, and 9 days with or without 1,25-(OH)2vitamin D3 (10(-8) M). In controls only small numbers of osteoclastic multinucleated cells 9MNCs) formed per well (< 15 per well). In contrast, 1,25-(OH)2D3 strongly stimulated MNC formation (> 80 per well on day 7). Messenger RNA (mRNA) for TRAP was detectable by reverse transcription-polymerase chain reaction amplification in both control and 1,25-(OH)2D3 treated groups at all times. However, TRAP mRNA was detectable in MNCs by the less sensitive in situ hybridization only on days 5, 7, and 9 and only in 1,25-(OH)2D3 treated cells. In control cultures, CTR mRNA was present on day 3 only in nonadherent cells and was not present in adherent cells (where MNCs formed) at any time point. In 1,25-(OH)2D3 treated cultures CTR mRNA was detectable in nonadherent cells on day 3 and in adherent cells on day 5 and thereafter. Peak levels of CTR mRNA were seen in adherent cells on day 7 (15-fold more than day 5 and 4-fold more than day 9). CT (10(-7) M) treatment of 7 day cultures, which had been stimulated to express the osteoclastic phenotype, caused a marked decrease in CTR mRNA expression at 24 h. There was no effect of CT treatment on CTR mRNA expression at 3 h or on TRAP mRNA expression at 3 or 24 h. In neonatal mouse calvaria cultures, CTR mRNA expression was constitutively present and was markedly decreased by 48 h of CT treatment. Similarly, bone resorption in these cultures was inhibited at 24 h by CT treatment, but at 48 and 72 h there was escape from the inhibitory effects of CT on resorption. In the marrow cultures, MNCs were greater than 98% positive for [125I]-salmon calcitonin (sCT) binding and this binding was completely competed away by excess cold sCT (10(-7) M). All primary isolated osteoclasts from 1- to 3-day-old mouse long bones exhibited [125I]-sCT binding and TRAP activity and were strongly positive for CTR and TRAP mRNA by in situ hybridization. Both MNCs that formed in bone marrow cultures and isolated primary osteoclasts formed resorption pits on bone slices.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1994

Murine osteoclasts and spleen cell polykaryons are distinguished by mRNA phenotyping.

To probe osteoclast gene expression, we combined the techniques of cell microisolation and RT-PCR to develop a novel and sensitive method for the isolation and mRNA phenotyping of small numbers of authentic osteoclasts and spleen cell polykaryons. Using this method we report (1) direct evidence for the presence of calcitonin receptor mRNA in osteoclasts, (2) confirmation of the recent finding of osteopontin mRNA in osteoclasts, and (3) demonstration that the specific expression of mRNA for tartrate-resistant acid phosphatase, carbonic anhydrase II, calcitonin receptor, and osteopontin enable one to distinguish the osteoclast from the morphologically similar and developmentally related spleen cell polykaryon. We also show that mRNA associated with the osteoblast phenotype, such as alkaline phosphatase, osteocalcin, and type I collagen, are absent in osteoclasts. This is the first report in which such an approach has been used successfully to distinguish the mRNA expression pattern of an authentic osteoclast from a macrophage polykaryon, and as such it should provide an important new tool for evaluating the results of various cell culture model systems designed to examine the origin and ontogeny of osteoclasts. Our results also indicate that these procedures can be used as an alternative to in situ hybridization methods for the cell-specific localization of specific mRNA in a mixed cell preparation and for colocalization of multiple mRNA species to a single cell type.

Animals↗
PubMed1994

Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene.

We have identified and characterized a mouse brain calcitonin receptor (CTR) complementary DNA (cDNA). This cDNA encodes a receptor protein that, after expression, has high affinity binding for salmon calcitonin (Kd approximately, 12.5 nM) and is coupled to adenylate cyclase. The binding affinity of this expressed receptor for salmon calcitonin is lower than that described for the previously cloned porcine renal and human ovarian CTRs, but is similar to that of the recently described rat brain CTR, designated the C1b form of the receptor. Analysis of the deduced structure of the mouse brain CTR reveals that it is highly related to the other CTR cDNAs that belong to a distinct family of G-protein-coupled receptors with seven transmembrane-spanning domains. The major structural feature that distinguishes the mouse cDNA clone from the other CTRs is the presence of a consecutive 111-basepair nucleotide sequence that encodes a 37-amino acid sequence which is predicted to localize to the first extracellular loop between the second and third transmembrane-spanning domains. We have mapped the CTR gene in the mouse to the proximal region of chromosome 6, which is homologous to the 7q region of human chromosome 7; only a single CTR gene was identified. Preliminary analysis of the mouse CTR gene reveals that it is complex, consisting of multiple exons separated by lengthy introns that would allow for splice variants consistent with the existence of multiple CTR isoforms predicted from the CTR cDNA clones. The differential cellular and tissue distribution of these functionally distinct CTR isoforms provides the molecular basis for the previously reported widespread distribution and functional heterogeneity of the CTR.

Amino Acid Sequence↗
PubMed1993

Characterization of the structural and functional properties of cloned calcitonin receptor cDNAs.

We have recently cloned CTRs from cDNA libraries prepared from porcine renal and human ovarian cell lines. In situ hybridization and Northern analysis confirm the widespread distribution of CTR mRNA in numerous tissues. Hydropathy plots of the predicted amino acid sequence of the receptors demonstrate multiple hydrophobic regions that could generate 7 transmembrane spanning domains, similar to other G protein-coupled receptors. Searches of databanks for proteins with related amino acid sequences reveals that the CTRs are closely related to the receptors for parathyroid hormone/parathyroid hormone related peptide, secretin, vasoactive intestinal peptide, growth hormone releasing hormone, glucagon-like peptide-1 and glucagon. These receptors have no significant sequence homology to other G protein-coupled receptors, and therefore, appear to comprise a distinct receptor family. Expression of the hCTR or pCTR in COS cells results in expression of high affinity CTRs which are coupled to adenylate cyclase (AC). The hCTR, however, demonstrates higher affinity for human and salmon CT compared to the pCTR. Both CTRs demonstrate low affinity binding and AC activation in response to calcitonin gene related peptide, amylin or secretin, providing a possible explanation for the cross-reactivity among these peptides in vivo. Stable transfectants expressing the pCTR increase cAMP levels and increases in cytosolic free Ca2+ concentration consistent with dual coupling to AC and phospholipase C. Additional studies will help to establish the structural basis for this functional property as well as the evolutionary relationship of the members of this newly identified family of receptors.

Animals↗
PubMed1993

Production of cytokines around loosened cemented acetabular components. Analysis with immunohistochemical techniques and in situ hybridization.

The chronic inflammatory response to wear particles from orthopaedic joint implants is believed to cause osteolysis and to contribute to prosthetic loosening. Previous in vitro experiments have demonstrated that particulate debris from joint implants causes cells in culture to release products that have been implicated in this pathological bone resorption. The purpose of the current study was to investigate the in vivo features of this complex process in patients who had had a total hip replacement. Membraneous tissue was obtained from the cement-bone interface of ten polyethylene acetabular components that had been revised for aseptic loosening in ten patients. The immunoperoxidase technique, which involves the use of specific antibodies for each cell type, showed that macrophages were the predominant cellular constituents but also that fibroblasts, many of which were not identified on plain histological study, were present and were actively producing collagen. T lymphocytes were present variably, but they generally composed less than 10 percent of the cells. Particulate debris (polyethylene, methylmethacrylate, and metal) was present in all membrane specimens but was intracellular only in macrophages and multinucleated giant cells. 35S-labeled nucleic-acid probes, complementary to human interleukin-1-beta and to platelet-derived growth-factor-2 messenger RNA (mRNA), were hybridized with serial tissue sections. Hybridization demonstrated interleukin-1-beta mRNA predominantly in macrophages, and not in fibroblasts or in T lymphocytes to any major extent. In contrast, immunolocalization demonstrated interleukin-1-beta protein on both macrophages and fibroblasts, suggesting that macrophages release interleukin-1-beta, which then binds to both fibroblasts and macrophages. Platelet-derived growth-factor transcripts were found in both macrophages and fibroblasts.

Aged↗
PubMed1992

A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C.

The signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.

Adenylyl Cyclases↗