Signal transduction by calcitonin

BRIEF REVIEWS and kidney. Although discussion of the biology of CT in this review is necessarily limited, an excellent in-depth treatment

Signal Transduction by Calcitonin Multiple Ligands, Receptors, and Signaling Pathways

can be found elsewhere (Azria 1989). CT or CT-like substances occur in a wide

William C. Horne, Jia-Fwu Shyu, Munmun Chakraborty, and Roland Baron

Calcitonin (CT) is a peptide hormone that is secreted by the parafollicular cells of the thyroid in response to elevated serum calcium levels. It acts to reduce serum calcium by inhibiting bone resorption and promoting renal calcium excretion. In addition to this hypocalcemic effect, calcitonin modulates the renal transport of water and several ions other than calcium and acts on the central nervous system to induce analgesia, anorexia, and gastric secretion. The CT receptor, a member of a newly described family of serpentine G protein-coupled receptors, has recently been shown to couple to multiple trimeric G proteins, thereby activating several signaling proteins, including protein kinase C, CAMP-dependent protein kinase and calcium/calmodulindependent protein kinase. In kidney proximal tubule cells (LLC-PIU), the CT-activated signaling mechanisms vary in a cell cycle-dependent manner, with the receptor coupling through a G, protein during G, phase and through a Gi protein and possibly a G, protein during S phase. These signaling mechanisms differentially modulate the activities of Na+/K+-ATPase and the apical Na+/H+ exchanger, effector molecules that play important roles in transepithelial Na+ transport. Cloning of CT receptors has revealed the presence of alternatively spliced cassettes, resulting in the expression of different isoforms of the receptor. The availability of these recombinant CT receptors has allowed preliminary characterization of the effects of changes in the receptor’s structure on its ligand binding and signal transduction properties. Thus, the cellular and molecular biology of CT is complex, with several structurally related peptide ligands and multiple isoforms of the CT receptor that can independently activate diverse signaling pathways. As the recent exciting results in this field are extended, we can expect rapid progress in understanding the molecular basis of the diverse effects of CT and, possibly, of the CT-related peptides CGRP and amylin. (Trends

Endocrinol Metab 1994;5:395-401)

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In mammals, the primary target organs for CT are bone and the kidney, where it lowers serum calcium concentrations by inhibiting osteoclast-mediated bone resorption and decreasing renal reabsorption of calcium (Azria 1989). These are not, however, the only effects or target organs of CT. In the kidney, CT also modulates the transport of other ions and water, exerting a potent natriuretic effect (Ardaillou 1975,

pocakemic activity (Copp et al. 1962), which results from its actions in bone

Bijvoet et al. 1971, Bidet et al. 1992, DiStefano et al. 1990). CT also affects the central nervous system, where it induces analgesia, gastric acid secretion, and inhibition of appetite (Fischer and Born 1987). Calcitonin is used therapeutically for treatment of a variety of disorders

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395

Calcitonin (CT), a 32-amino-acid William C. Home, Jia-Fwu Shyu, Munmun Chakraborty, and Roland Baron are at the Departments of Cell Biology and Orthopaedies, Yale University School of Medicine, New Haven, CT 06520-8044, USA.

range of organisms, from unicellular species to mammals. The primary structures of CT from eight species (human, bovine, pig, sheep, rat, chicken, salmon, and eel) have been determined. Six of the first nine residues at the N-terminal are invariant (seven in all species other than chicken), with a disulfide bond between Cyst and Cys, forming a characteristic ring structure. Gly,, and a C-terminal proline amide group are also present in all eight species. The central regions of the peptides are more divergent, although even in this portion of the molecule there are high degrees of identity within groups of two or three species. Thus, human and rat CT are identical except at positions 16 and 26, eel CT differs from salmon and chicken CT by three and two residues, respectively, and bovine, sheep, and pig CT are closely related. These structural differences are clearly of functional significance, because the relative affinities of the different CTs for CT receptors, determined by direct binding and cellular response, differ by lo-fold or more, with salmon CT binding with a higher affinity than mammalian CTs to CT receptors of all species, even when the mammalian CT and CT receptor are from the same species .

hormone

secreted by the parafollicular

or C cells originally

peptide

of the thyroid identified

based

gland, on

was

its hy-

involving hypercalcemia and/or elevated bone resorption, such as hypercalcemia of malignancy, osteoporosis, and Paget’s disease, and has also been used for its analgesic properties (Azria 1989). In addition to CT itself having several target organs and diverse effects, the potential complexity of the CT system is further increased by the existence of at least two other structurally related endocrine or paracrine peptides. These are calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide that is also encoded by the CT gene and arises from alternative splicing of the initial CT gene transcript, and amylin, a 37-aminoacid peptide encoded by a gene that appears to have evolved from the same ancestral gene as the CT/CGRP gene [reviewed in Fischer and Born (1987) and Zaidi et al. (1990b)l. The Nterminal domains of CGRP and amylin form disulfide-bonded ring structures similar to that of CT, but the two peptides are otherwise quite dissimilar to CT (16% homology between human CT and human CGRP, 20% homology between amylin and salmon CT). CGRP is predominantly synthesized in the central and peripheral nervous system, although it is present in and secreted by the thyroid. The presence of CGRP in peripheral nerves that are associated with blood vessels reflects its activity as a vasodilator. It also enhances skeletal muscle contraction and induces increased rate and force of cardiac contraction (Fischer and Born 1987). In the kidney, it increases blood flow and relaxes mesangial cells-effects that are independent of CT receptors-and modulates water and electrolyte transport in a manner similar to CT, probably by binding to CT receptors (Kurtz et al. 1989, Wohlwend et al. 1985). CGRP has also been recently reported to affect the secretion of interleukin-2 by T lymphocytes (Wang et al. 1992). Amylin is secreted by pancreatic fi cells. Its primary effect is in the regulation of carbohydrate metabolism, particularly in muscle, where it inhibits glucose uptake and glycogen synthesis. Both CGRP and amylin have hypocalcemic effects when administered pharmacologically and inhibit osteoclasts in vitro, probably owing to binding to CT receptors, both on osteoclasts and in the kidney (Zaidi et al. 199Ob). 3%

??

Physiologic Roles of Calcitonin

The hypocalcemic effect of CT is primarily due to its profound inhibitory action on osteoclastic bone resorption, with some additional contribution from increased excretion of Ca2+ by the kidney. In bone, actively resorbing osteoclasts seal off an area of bone surface and secrete acid and a variety of acid hydrolases into the resulting space between the cell and the bone via the ruffled border, a specialized, highly invaginated membrane structure that is enriched in vacuolar-type proton pumps and contains a number of proteins characteristic of the endosomal/lysosomal pathway [reviewed in Baron et al. (1993)]. Administration of CT induces the rapid internalization of the ruffled border proteins into small intracellular vesicles (Baron et al. 1990), thereby halting acid secretion and the resulting demineralization of the bone matrix. In isolated osteoclasts, CT causes reduced osteoclast motility (the Q effect) and retraction of the osteoclast from its usual spread morphology (the R effect) and, possibly as a result of one or both of these activities, inhibits bone resorption as measured by the formation of pits in bone slices (Zaidi et al. 1990a, Su et al. 1992). In the kidney, CT enhances the fractional excretion of sodium, chloride, potassium, calcium, and phosphate and stimulates urine flow, as well as influencing the metabolism of vitamin D (Kurokawa et al. 1992). Whereas species differences in the specific effects of CT make the integration of results from different studies problematic, it seems clear that CT acts at a variety of sites throughout the kidney. Thus, CT’s natriuretie effect is attributed mostly to an inhibition of sodium reabsorption in the proximal tubule (Giebisch and Aronson 1986), and CT-induced changes in phosphate transport (Berndt and Knox 1984) and vitamin D metabolism (Kawashima et al. 1981) in the rat proximal tubule have also been reported. On the other hand, studies in isolated rabbit and human nephrons have shown that CT activates adenylate cyclase in the medullary and cortical thick ascending limb of the loop of Her-de and the distal convoluted tubule, but not in the proximal tubule (Chabardes et al. 1976 and 1980, Murphy et al. 1986). The distribution of both immunoreactive CT-like substances (iCT) and CT 01994, Elsevier Science Inc., 1043-276Ol94l$7.00

receptors in human and rat brain has been characterized. Binding sites for 1251-salmon CT are present in the pituitary and several regions of the diencephalon and the brain stem, including the hypothalamus and the reticular formation (Sexton 1992). The distribution of iCT is in part congruent with that of the binding sites, with the highest concentrations found in hypothalamus, median eminence, and pituitary (Sexton 1992). The well-defined distribution of iCT and CT receptors in the central nervous system implies that CT and/or CT-like peptides play specific physiologic roles there. Although such functions have not yet been well characterized, the presence of high levels of both iCT and CT receptors in the hypothalamus-a region of the brain that is involved in pain recognition, feeding, and the regulation ofpituitary hormone secretion-suggests that CT’s antinociceptive and analgesic effects are the results of direct effects of the hormone on cells in this area. CT thus acts at multiple sites to induce a variety of cell-specific effects. The diverse nature of the cellular responses, as well as the apparent discrepancies between the patterns of physiologic response and activation of adenylate cyclase in the kidney, highlights the need to better characterize CT-activated signal transduction in the various target cells and to evaluate the possibility that the presence of multiple CT receptors might underlie the observed physiologic diversity.

??

Calcitoniu Activates Multiple Signal Transduction Pathways

Although early investigations of CTactivated signal transduction focused on adenylate cyclase, it is now clear that CT receptors couple to several independent signaling pathways. Studies in our laboratory, using the kidney proximal tubule cell line LLC-PKl, have shown that the CT receptor signals through multiple G protein-coupled pathways (Figure 1) (Chakraborty et al. 1991 and 1994). Interestingly, the mode of receptor coupling and the biologic effects of CT vary with the stage of the cell cycle in synchronized cells. In the G, phase, CT activates adenylate cyclase, coupling through a G, protein, with the resulting sixfold increase in CAMP levels causing a two- to threefold increase in ouabain

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binding to the plasma membrane Na+/K+ATPase.

This

effect

of CT on ouabain

binding is mimicked by agents known to activate adenylate cyclase (forskolin, isoproterenol,

cholera

toxin) as well as by

CAMP analogues and is insensitive to a variety of agents (pertussis toxin, phorbol esters, sphingosine) that activate or inhibit other G protein-coupled signaling mechanisms. A completely different situation occurs during S phase, where CT treatment has little effect on CAMP levels, and ouabain binding is markedly decreased. This effect on ouabain binding can be mimicked by phorbol esters and inhibited by sphingosine, suggesting the involvement of protein kinase C (PKC). CT also activates calcium/calmodulin-dependent protein kinase II (CaM kinase II) in S phase cells, because a CT-induced inhibition of 22Na uptake is blocked by CaM kinase II-specific inhibitory peptides (Chakraborty et al. 1994). Rather than coupling to the cholera toxin-sensitive G, protein, as it does during G, phase, the CT receptor couples to a Gi protein, as indicated by the reversal of the CT-induced decrease in ouabain binding by pertussis toxin. Coupling to Gi has been reported to decrease the affinity of other G protein-coupled receptors for their respective ligands, and, indeed, the affinity of the receptor for CT also changes in S phase, decreasing from 1.8 nM to 3.2 nM. Receptorinduced activation of the Gi protein could be responsible for all the CTrelated signaling phenomena detected during the S phase of the cell cycle, because the a, subunit inhibits adenylate cyclase, and l3y subunit complexes can activate phospholipase Cl3 (PLCfi (Clapham and Neer 1993) with the subsequent generation of diacyl glycerol, a PKC activator, and inositol trisphosphate (IP,), which triggers the release of sequestered Ca2+ from intracellular stores, leading to the activation of CaM kinase II. The activation of PLC by a G, a subunit rather than the Gi @y subunits has not, however, been ruled out. Evidence from a variety of other systems supports the idea that the CT receptor can couple to both adenylate cyclase and PLC. In osteoclasts, CT induces increases in both CAMP and intracellular calcium ([Ca2+li) , and the inhibitory effect of CT on bone resorption can be mimicked by either CAMP analogues or calcium ionophores (Zaidi

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ATPase Figure 1. Calcitonin (CT)-induced signal transduction in synchronized LLC-PKl cells. In G2 phase, the CT receptor couples to a G, trimeric G protein to activate adenylate cyclase (AC), elevating CAMP levels and activating CAMP-dependent protein kinase (PKA), which subsequently induces an increase in the activity and/ornumber of plasma membrane Na+/K+-ATPase, as measured by ouabain binding to intact cells. During S phase, the CT receptor couples to a G, trimeric G protein, inactivating adenylate cyclase. Phospholipase C (PLC) is also activated, by either the Gi & subunits or by a G, trimeric G protein, generating diacyl glycerol and inositol trisphosphate, which activate protein kinase C (PKC) and increase cytosolic Ca*+ ([Ca*+],) levels, respectively. Increased [Ca*+J activates calcium/caimoduiin-dependent protein kinase (CaM PIUI), which inhibits the apical Na+/II+antiporter (NI-IE Ap). The resulting reduction in cytosolic [Na+] in turn leads to decreased Na+/K+-ATPaseactivity. PKC also appears to inhibit the Na+/K+-ATPase,in a CaM PIUI-independent manner.

et al. 1990a, Alam et a.l.1991, Su et al. 1992, Moonga et al. 1992, Malgaroli et al. 1989). Furthermore, PKC also appears to be involved, because inhibitors of this enzyme partially block the effect

ment with ionomycin or pertussis toxin (Zaidi et al. 199Oa) and blocked by inhibitors of PKC (Su et al. 1992). Interestingly, the two CT-related peptides, CGRP and amylin, induce the Q

of CT on bone resorption,

effect but fail to elicit either

and phorbol

esters, which activate PKC, inhibit bone resorption (Su et al. 1992). The R and Q effects appear to be mediated by different signaling mechanisms. Both dibutyryl CAMP and cholera toxin induce the Q effect but not the R effect, whereas the R effect is specifically induced by treat-

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a rise in

[Ca2+], or the resulting R effect (Alam et al. 1993). In the kidney, CT activates both the adenylate cyclase and PLC signaling pathways, although in distinctly different regions of the nephron. CT-dependent activation of adenylate cyclase occurs in the medullary and

397

cortical thick ascending limb and the distal convoluted tubule, where it promotes Ca2+ reabsorption, but not in the proximal tubule (Chabardes et al. 1976 and 1980, Murphy et al. 1986). In contrast, increases in [Ca2+li, presumably reflecting the activation of PLC and the resulting generation of IP,, are seen in proximal straight tubules and in medullary thick ascending loops (Murphy et al. 1986, Suzuki et al. 1989). The existence of another as yet uncharacterized coupling mechanism is implied by studies of CT signaling in brain membrane preparations. Here, in contrast to its effect in bone and kidney cells, CT appears to inhibit adenylate cyclase (Rizzo and Goltzman 198 1, Nicosia et al. 1986). This inhibition is apparently independent of inhibitory G proteins, however, because pertussis toxin, which interferes with Gi signaling, does not block the CT-induced inhibition of brain adenylate cyclase activity (Guidobono et al. 1991). Further investigation will therefore be required to fully elucidate the mechanism(s) by which CT acts in the brain. we have begun to identify some of the downstream effector proteins in the LLCPKl system whose activities are regulated in response to CT (Chakraborty et al. 1991 and 1994) (Figure 1). As noted earlier, CT has marked effects on ouabain binding to LLC-PKl cells, reflecting changes in the number and/or activity of the Na+/K+-ATPasemolecules expressed at the cell surface. A second ion transporter whose activity changes in response to CT treatment is the apical Na+/H+exchanger (NHE), which is markedly inhibited in CT-treated S phase cells via a mechanism that involves CaM kinase II. Na+ reabsorption in the kidney results mostly from the activity of the apical NHE and the Na+/K+-ATPase (Giebisch and Aronson 1986). Thus, the potent inhibition of these Na+-transporting proteins by CT provides a likely mechanism for the natriuretic effect of CT in vivo. Regulation of these or similar proteins in other target cells could be expected to result in altered cellular function. In osteoclasts, although the apical form of the NHE is probably not expressed, Na+/K+-ATPase and NHE activities are required for the maintenance of intracellular pH and transmembrane electrical potential during the periods of active 398

acid secretion required for bone resorption. Their inhibition would therefore likely lead to changes in intracellular electrolyte balances that are incompatible with bone resorption, as well as in changes in Na+-mediated cell volume regulation, thus contributing to CT’s inhibitory effect on osteoclast activity. Similarly, inhibition of Na+ transporters, particularly the Na+/K+-ATPase,in excitatory cells of the nervous system would have profound effects on neuronal activity, if CT can indeed regulate the Na+/K+ATPase in this cellular environment. The effects of CT are, however, certainly not limited to ion transporters. The kinases that are activated in response to CT (PKA, PKC, CaM kinase II) contribute to the regulation of multiple cellular functions, including endocytosis and exocytosis, cellular motility, and adhesion to extracellular matrix, that are directly involved in the physiologic functions of the cells that respond to CT Further studies will undoubtedly reveal numerous specific intracellular effecters that mediate the action of CT on cellular activity.

??

Cloning and Characterization of Calcitonin Receptors

Our current understanding of the physiology and pharmacology of CT and CT-like peptides such as CGRP and amylin therefore raises several questions about the nature and structural relationships of the receptors for this family of peptides. The target cells of each peptide and the nature of their responses clearly indicate that there are different receptors that effectively discriminate between the individual peptides (for example, on lymphocytes or on mesangial cells, where CGRP but not CT induces cellular responses) as well as receptors that may bind and respond to more than one of the family members, albeit with strikingly different affinities (for example, on renal epithelial cells and osteoclasts). Investigation of the structural determinants of CT activity provides further support for the existence of multiple receptor subtypes. In particular, there are clear differences in the abilities of receptors in brain, kidney, and osteoclast to bind and respond to analogues of salmon CT that lack the ability of the native peptide to form an amphipathic helix (Twery et al. 1988a 01994, Elsevier Science Inc., 1043-2760/94/$7.00

and b, Nakamuta et al. 1990). However, much work remains to be done to fully characterize these receptors in terms of structure, physiologic role, and pharmacologic properties. The activation of multiple signal transduction mechanisms by CT also raises the possibility that there may be multiple receptor subtypes. Although many of the G protein-coupled receptors apparently couple to only one signaling pathway, there are examples of receptors that activate more than one signaling mechanism (Clapham and Neer 1993). Are there, therefore, specific CT receptor subtypes that couple to one or another of the intracellular signal transducers, or is there a single CT receptor that activates multiple signaling pathways? If a single receptor in fact couples to both adenylate cyclase and PLC, then the cell cyclespecific pattern of signaling that we have described in LLC-PKl cells and the apparently selective activation of different signaling pathways in different regions of the nephron suggest that the coupling must be regulated, but the nature of such regulation remains unknown. The recent isolation and characterization of cDNAs encoding CT receptors from pig (Lin et al. 1991) human (Gorn et al. 1992, Moore et al. 1992), rat (Albrandt et al. 1993, Sexton et al. 1993) and rabbit (Shyu et al., unpublished observations) provides the opportunity to begin to examine these questions at the molecular level. The receptors show about 6O%-80% identity at the amino acid level among the species. Analysis of the deduced CTR amino acid sequences reveals the presence of seven hydrophobic regions that are predicted to be membrane-spanning domains, indicating that the CTR is indeed a member of the seven-transmembrane, or serpentine, G protein-linked receptor superfamily, as was expected from the signaling pathways that are activated by CT. The most striking regions of sequence conservation among the different CTRs lie in the putative membrane-spanning domains. The CTR cDNA sequences have no significant sequence identity (< 12%) with other more classical G proteincoupled receptors such as the adrenergic, muscarinic and dopaminergic receptors. They are, however, more closely related (25%50% identity) to other recently cloned cDNAs that encode recep-

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tom

for

a variety

of

small

peptide

hormones, including parathyroid hormone/parathyroid hormone-related peptide (PTIWPTHrP), vasoactive

secretin, glucagon,

intestinal

peptide

(VIP),

glucagonlike peptide 1 (GLP-l), and GHRH, which define a new family of G protein-coupled receptors [for review, see Segre and Goldring (1993)]. The close relationship of these receptors extends to the organization of the genes. In contrast to many of the more classical G protein-coupled receptors, which have few or no introns, the genes that encode the receptors for CT, PTH/PTHrP and GHRH, which have only recently been cloned and characterized, have 12 to 15 exons, arranged in a closely related manner (Zolnierowicz et al. 1994, Lin et al. 1993, McCuaig et al. 1994). Some of the functional properties of the recombinant receptors have been characterized. Most interesting for this discussion, when the recombinant porcine receptor is expressed in cells that normally do not respond to calcitonin, treatment with the hormone leads to the activation of both adenylate cyclase and phospholipase C (Chabre et al. 1992, Force et al. 1992). Thus, the coupling of the CTR to multiple G proteins that was found in kidney cells (Chakraborty et al. 1991 and 1994) and osteoclasts (Zaidi et al. 1990a, Su et al. 1992) is not necessarily due to the presence of different isoforms that bind calcitonin but couple to different signaling pathways. As noted earlier, however, instances of independent activation of some subset of these signal transduction mechanisms require that the coupling be a regulated phenomenon, possibly involving phosphorylation of the receptor. Interestingly, some of the other receptors that are most closely related to the CTR (PTH/PTHrP, glucagon, and GLP-1 receptors) also couple to more than one G protein (Segre and Goldring 1993) suggesting that this may be a characteristic property of this family of receptors. Although all four species (pig, human, rat, and rabbit) express mRNAs that encode receptors homologous to the originally cloned porcine receptor (Lin et al. 199 1, Moore et al. 1992, Albrandt et al. 1993, Sexton et al. 1993, Shyu unpublished observations), additional isoforms that apparently arise from the presence of alternatively spliced cassettes have been also been identified in

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human, rat, and pig cells or tissues. Thus, it is clear that multiple receptor isoforms species.

can be expressed

in the same

Two

alternatively

additional

spliced forms have been identified (Figure 2). One, which contains a 16-amino-acid sequence in the putative first intracellular loop [found in both human and pig cells (Gom et al. 1992, Zolnierowicz et al. 1994)], is the result of splicing exon 7 to one or the other of two 3’ splice acceptor sites at the 5’ end of exon 8 (Zolnierowicz et al. 1994). The second, which contains a 3i’-amino-acid sequence in the putative first extracellular loop [found in a rat brain cDNA (Albrandt et al. 1993, Sexton et al. 1993)], may be the result of an alternatively spliced exon (Zolnierowicz et al. 1994). Although only these two alternatively spliced cassettes have been identified at this time, the presence of at least 14 exons in the CT receptor gene (Figure 3) (Zolnierowicz et al. 1994) makes it likely that a rigorous analysis of CT receptor mRNA from various tissue sources will reveal the presence of other such cassettes. Although the functional differences of these CTR isoforms are not yet established, the different receptors might be expected to exhibit altered ligand binding or receptor-G protein interactions, by analogy with other alternatively spliced G protein-coupled receptors (Namba et al. 1993, Spengler et al. 1993). In fact, the two rat isoforms, which differ only in the presence of the insert in the first extracellular loop, have distinctly different relative affinities for salmon, porcine, and human CT (Sexton et al. 1993). In addition, Gom and colleagues have suggested that the 16-amino-acid insert in the first intracellular loop of

out

nn

In

Figure 2. Model of the CT receptor. The locations of the two identified alternatively spliced cassettes are indicated.

their cloned human CTR might be responsible in part for the differences in the ligand binding affinities of the recombinant human and porcine CTR (Gom et al. 1992). In this case, however, there are also a number of differences in the amino acid sequences of the two proteins, particularly in the extracellular N-terminal domains, which might also contribute to differences in ligandreceptor interactions. Alternatively spliced isoforms might even have completely different ligand specificities. Although a variety of studies indicate that CT, CGRP, and amylin act through different receptors, the three peptides will bind to each other’s receptors at high ligand concentration (Zaidi et al. 1990b). Because the receptors for CGRP and amylin have not yet been characterized at the structural level, it is possible that alternative splicing of the transcripts of a single CTR gene could lead to the synthesis of related receptor

Figure 3. Structure of the porcine CTR gene. The approximate positions of the 14 exons are shown in the drawing of the gene. The sites of the exon boundaries are indicated relative to the mRNA sequence. The positions in the mRNA of the sequences that encode the seven predicted transmembrane domains (I-VII) of the receptor protein are indicated in white. Adapted from Zolnierowicz et al. (1994).

Gene

5’

mRNA

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399

proteins with different relative affinities for the structurally related peptide ligands. Recently, Sexton and colleagues have provided data that support this hypothesis and, in addition, demonstrate that some of the identified CT receptor isoforms may respond specifically to other hitherto unidentified CT-like peptides. They show that one of the two CT receptor isoforms cloned from rat brain (Clb, which contains the 37-amino-acid insert in the first extracelular loop) fails to bind human or rat CT or to mediate activation of adenylate cyclase at CT concentrations as great as lti M, suggesting that this isoform does not respond to endogenous CT in the rat. Furthermore, neither CGRP nor amylin bind to the Clb receptor. On the other hand, salmon CT does bind to Clb and induces the activation of adenylate cyclase, showing that it is a functional receptor (Sexton et al. 1993). Sexton suggests that the ligand for the Clb isoform may therefore be a recently identified peptide from rat brain that is biologically, chromatographically, and immunologically similar to salmon CT and can be immunologically distinguished from mammalian CT and CGRP (Sexton and Hilton 1992). It thus appears that the presence of multiple isoforms of the CT receptor indeed may account for some of the complexity of the physiologic and pharmacologic responses to CT and the other CT-like peptides. ??

Conclusions

Although CT was originally described as a factor that exerted a hypocalcemic effect in response to elevated serum calcium, it is now clear that this hormone has multiple physiologically important effects on tissues as different as bone, kidney, and brain. Recent advances in elucidating the cellular physiology and molecular biology of CT and other CTrelated peptides have demonstrated a previously unsuspected degree of complexity in the number of structurally related ligands, in the receptors for these ligands, and in the nature of signaling mechanisms to which the receptors couple. The high degree of similarity of the sequences of the different cloned CTR cDNAs, particularly in the transmembrane regions, will permit the design and construction of chimeric recombinant receptors, thereby facilitating the eluci-

400

dation of the roles of specific alternatively spliced cassettes in determining the specificity of ligand binding and G protein coupling of different CT receptors. There is clearly much more to learn about how these peptides and their receptors act, and we can undoubtedly expect rapid progress as molecular biologic techniques are applied to these investigations.

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Albrandt K, Mull E, Brady EMG, Herich J, Moore CX, Beaumont K: 1993. Molecular cloning of two receptors from rat brain with high affinity for salmon calcitonin. FEBS Lett 325:225-232.

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Azria M: The Calcitonins: Physiology and Pharmacology. Basel, Karger, 1989. Baron R, Neff L, Brown W, Louvard D, Courtoy PJ: 1990. Selective internalization of the apical plasma membrane and rapid redistribution of lysosomal enzymes and mannose 6-phosphate receptors during osteoclast inactivation by calcitonin. J Cell Sci 97439-447. Baron R, Ravesloot J-H, Neff L, et al.: 1993. Cellular and molecular biology of the osteoclast. In Noda M, ed. Cellular and Molecular Biology of Bone. Orlando, FL, Academic Press, pp 445-495.

Fischer JA, Born W: 1987. Calcitonin gene products: evolution, expression and biological targets. Bone Miner 2:347-359. Force T, Bonventre JV, Flannery MR, Gom AH, Yamin M, Goldring SR: 1992. A cloned porcine renal calcitonin receptor couples to adenylate cyclase and phospholipase C. Am JPhysiol262:FlllO-F1115.

Bemdt TJ, Knox FG: 1984. Proximal tubule site of inhibition of phosphate reabsorption by calcitonin. Am J Physiol246:F927-F930.

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Bidet M, Taut M, Gastineau M, Poujeol P: 1992. Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule. Pfltigers Arch 421:523-529.

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Kurokawa K, Fukagawa M, Hayashi M, Sax-&a T: 1992. Renal receptors and cellular mechanisms of hormone action in the kidney. In Seldon DW, Giebisch G, eds. The Kidney: Physiology and Pathophysiology. New York, Raven, pp 1339-1372. Kurtz A, Muff R, Fischer JA: 1989. Calcitonin gene products and the kidney. Klin Wochenschr 67~870-875. Lin HY, Harris TL, Flannery MS, et al.: 1991. Expression cloning of an adenylate cyclasecoupled calcitonin receptor. Science 254:1022-1024. Lin S-C, Lin CR, Gukovsky I, Lusis AJ, Sawchenko PE, Rosenfeld MG: 1993. Molecular basis of the little mouse phenotype and implications for cell type-specific growth. Nature 364:208-213. Malgaroli A, Meldolesi J, Zambonin Zallone A, Teti A: 1989. Control of cytosolic free calcium in rat and chicken osteoclasts: the role of extracellular calcium and calcitonin. J Biol Chem 264:14,342-14,347. McCuaig KA, Clarke JC, White JH: 1994. Molecular cloning of the gene encoding the mouse parathyroid hormone/pamthyroid hormone-related peptide receptor. Proc Nat1 Acad Sci USA 91:5051-5055. Moonga BS, Alam ASMT, Bevis PJR, et al.: 1992. Regulation of cytosolic free calcium in isolated rat osteoclasts by calcitonin. J Endocrinol 1321241-249. Moore EE, Kuestner RE, Thompson DL, et al.: 1992. The cloned human calcitonin receptor can couple to two alternative second messenger pathways when expressed in BHK cells [abst]. J Bone Miner Res 7(Suppl l):S146.

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Do you have ideas for articles you would like to see in future issues of

Murphy E, Chamberlin M, Mandel LJ: 1986. Effects of calcitonin on cytosolic Ca in a suspension of rabbit medullary thick ascending limb tubules. Am J Physic)125l:C491c495. Nakamuta H, Orlowski RC, Epand RM: 1990. Evidence for calcitonin receptor heterogeneity: binding studies with nonhelical analogues. Endocrinology 127: 163-l 69. Namba T, Sugimoto Y, Negishi M, et al.: 1993. Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity. Nature 365:166-170.

If so, please send your suggestionsfor topic(s) and author(s) to:

Nicosia S, Guidobono F, Musanti M, Pecile A: 1986. Inhibitory effects of calcitonin on adenylate cyclase activity in different rat brain areas. Life Sci 39:2253-2262.

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Rizzo AJ, Goltzman D: 1981. Calcitonin receptors in the central nervous system of the rat. Endocrinology 108:1672-1677. Segre GV, Goldring SR: 1993. Receptors for secretin, calcitonin, parathyroid hormone (PTH)/PTH-related peptide, vasoactive in-

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