Biomedical subjects

P M Sexton

Publications and source records attributed to P M Sexton.

At least 19 recordsLinked to original sources

PubMed2002

Molecular pharmacology of the calcitonin receptor.

The peptide hormone calcitonin is widely used therapeutically in the treatment of bone disorders such as Paget's disease, osteoporosis, and the hypercalcemia of some malignancies. However, emerging evidence suggests the actions of calcitonin via its G protein-coupled receptor, the calcitonin receptor, may not be limited to bone. Calcitonin receptors have also been identified in the central nervous system, testes, skeletal muscle, lymphocytes, and the placenta. We are now becoming aware that the complexity of the calcitonin response mediated by the calcitonin receptor can be influenced by accessory proteins, receptor isoforms, genetic polymorphisms, developmental and/or transcriptional regulation, feedback inhibition, and the specific cellular or tissue background. This article discusses what is known about the molecular and pharmacological actions of the calcitonin receptor and highlights areas of current research.

Alternative Splicing↗

PubMed2001

Receptor activity modifying proteins.

Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines our current state of knowledge of the mechanisms underlying RAMP function.

Amino Acid Sequence↗

PubMed2000

Mouse receptor-activity-modifying proteins 1, -2 and -3: amino acid sequence, expression and function.

The calcitonin receptor-like receptor (CRLR) requires novel receptor-activity-modifying proteins (RAMPs) for its function as an adrenomedullin (ADM) or a calcitonin (CT) gene-related peptide (CGRP) receptor. Here, mouse cDNA clones representing expressed sequence tags (ESTs) in the GenEMBL database have been identified. They encode for proteins with 70, 68 and 84% amino acid sequence identity with respect to human RAMP1, -2 and -3. On Northern blot analysis of polyA(+) RNA mouse RAMP1 (mRAMP1) encoding mRNA with an apparent size of 0.8 kb was predominantly observed in embryonic and adult brain and lung and in adult skeletal muscle. Mouse RAMP2 encoding 0.8 and 1.2 kb mRNA were recognized in all tissues analyzed with the highest levels in embryonic brain, lung and gut and in adult heart, lung, skeletal muscle and brain. A single 1.2 kb mRAMP3 encoding transcript was mainly expressed in embryonic and adult brain. In COS-7 cells co-expressing rat CRLR (rCRLR) and mRAMP1, [125I]halphaCGRP binding was inhibited by ralphaCGRP(8-37), ralphaCGRP and rbetaCGRP with IC(50) of 1.4+/-0.5, 4.5+/-0.6 and 7+/-0.3 nM, respectively. CyclicAMP accumulation was maximally stimulated tenfold by rbetaCGRP and ralphaCGRP with EC(50) of 0. 65+/-0.67 and 0.86+/-0.6 nM. In the same cells co-expressing rCRLR and mRAMP2, binding of [125I]rADM was displaced by rADM and rADM(20-50) with IC(50) of 1.9+/-0.5 and 3.4+/-1.4 nM, respectively, and a maximal sevenfold stimulation of cAMP accumulation was observed with rADM with an EC(50) of 0.82+/-0.85 nM. In the cells co-expressing rCRLR and mRAMP3, [125I]halphaCGRP binding was inhibited by ralphaCGRP(8-37), rbetaCGRP, ralphaCGRP, rADM and rADM(20-50) with IC(50) between 4 and 22 nM. cAMP accumulation was stimulated by rADM with an EC(50) of 5.1+/-2.7 nM that was 12-fold and 11-fold lower than that of ralphaCGRP and rbetaCGRP. In conclusion, mouse RAMP1, -2 and -3 exhibit high amino acid sequence homology to the corresponding human RAMPs. Co-expression of rCRLR with mRAMP1, -2 or -3 in COS-7 cells revealed distinct CGRP-, ADM- or ADM/CGRP receptors.

Adrenomedullin↗

PubMed2000

Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product.

Calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin, and adrenomedullin constitute a family of structurally related peptides that signal via either the calcitonin receptor-like receptor or the CT receptor, with receptor phenotype determined by coexpression of one of the three receptor activity-modifying proteins (RAMPs). The nature of the interaction between the receptor and RAMP was investigated using chimeras between RAMP1 and RAMP2 where the amino-terminal domain of RAMP1 was attached to the transmembrane domain and carboxy terminus of RAMP2 and called RAMP1/2, and vice versa for RAMP2/1. Cotransfection of wild-type or chimeric RAMPs with the insert-negative isoform of the human CT receptor (hCTR(I1-)) into COS-7 cells resulted in the expression of (125)I-rat amylin binding sites. Highest specific binding was observed when either RAMP1 or RAMP2/1 were cotransfected, indicating the importance of the RAMP transmembrane domain and/or carboxy terminus for the degree to which amylin receptors are expressed. In contrast, the phenotype generated was primarily determined by the amino terminus, with similar RAMP1- and RAMP1/2-induced receptor phenotypes that had higher affinity for human CGRPalpha and lower affinity for human calcitonin than the RAMP2- and RAMP2/1-induced receptors.

Amyloid↗

PubMed2000

Clozapine and risperidone treatment of psychosis in Parkinson's disease.

The authors compared efficacy and safety of risperidone and clozapine for the treatment of psychosis in a double-blind trial with 10 subjects with Parkinson's disease (PD) and psychosis. Mean improvement in the Brief Psychiatric Rating Scale psychosis score was similar in the clozapine and the risperidone groups (P=0.23). Although the mean motor Unified Parkinson's Disease Rating Scale score worsened in the risperidone group and improved in the clozapine group, this difference did not reach statistical significance. One subject on clozapine developed neutropenia. In subjects with PD, risperidone may be considered as an alternative to clozapine because it is as effective for the treatment of psychoses without the hematologic, antimuscarinic, and seizure side effects. However, risperidone may worsen extrapyramidal symptoms more than clozapine and therefore must be used with caution.

Aged↗

PubMed2000

Identification of key components in the irreversibility of salmon calcitonin binding to calcitonin receptors.

This study investigates the poor reversibility of salmon calcitonin (sCT) binding to rat and human calcitonin receptors. Efficacy of CT and analogue peptides in (125)I-sCT binding competition and cAMP assays was compared with the dissociation kinetics of (125)I-labelled peptides. Assessment was performed on cells stably expressing either rat or human calcitonin receptors. Dissociation kinetics of the antagonists, sCT(8-32) and AC512, revealed that binding was rapidly and completely reversible at the receptors, despite high affinity binding, suggesting that poor reversibility required the active conformation of the receptor. G protein coupling was not essential as the dissociation kinetics of (125)I-sCT binding to cell membranes did not significantly alter in the presence of GTP gamma S. Time course experiments established that the transition to irreversibility was slow, while the reversible component of binding appeared to involve a single population of either receptor states or binding sites. Pre-bound (125)I-human CT dissociated rapidly from the receptors, indicating that not all agonists bound irreversibly. To identify structural features of sCT that contribute to its poor reversibility, dissociation kinetics of sCT analogues with various structural modifications were examined. Increasing truncation of N-terminal residues of sCT analogues led to a corresponding increase in the rate of peptide dissociation. Salmon CT peptides which had been substituted at the N-terminus by 13-21 residues of human CT (hCT) were equipotent with sCT in binding competition and cAMP accumulation assays but exhibited a dissociation rate similar to hCT. In contrast, despite lower affinity and efficacy at the receptors, the chimeric analogue sCT(1-16)-hCT(17-32) displayed poorly reversible binding, similar to sCT. Analysis of the dissociation kinetics of sCT analogues with differing alpha-helix forming potential indicated that the ability to form alpha-helical secondary structure was an important factor in the rate of ligand dissociation. We hypothesise that poor reversibility results from a conformational change in the receptor and/or ligand and that this is dependent, at least in part, on interaction with residues constrained within the alpha-helix of the peptide.

Animals↗

PubMed2000

Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment.

Receptor activity modifying proteins (RAMPs) constitute a group of three proteins, designated as RAMP1, 2, and 3, which are able to effect functional changes in some members of the G protein-coupled receptor family. Thus, RAMP1 or RAMP3 can modify the calcitonin receptor (CTR) to also function as a high-affinity amylin receptor-like phenotype. To examine the RAMP/CTR interaction, individual RAMPs were coexpressed with either of the two human CTR (hCTR) isoforms, the insert negative (hCTR(I1-)) or the insert positive (hCTR(I1+)), in Chinese hamster ovary (CHO-P) or African monkey kidney (COS-7) cells. CHO-P cells provide an environment conducive to a low, but significant, level of amylin binding with either hCTR isoform alone, unlike in COS-7, where RAMP coexpression is imperative for amylin binding. Also, in CHO-P, hCTR(I1-) induced amylin binding with all three RAMPs, in contrast to COS-7, where only RAMP1 or RAMP3 generate an amylin receptor phenotype. hCTR(I1+) induced high-affinity amylin binding with any RAMP in either cell line. In COS-7 cells, hCTR(I1+)/RAMP-generated receptor displayed high- and low-affinity states, in contrast with the single-state binding seen with hCTR(I1-)/RAMP-generated receptor, whereas in CHO-P cells a two-affinity state receptor phenotype was evident with both hCTR isoforms. Endogenous RAMP expression is low and similar between cell lines. The results suggest that CTR/RAMP interaction in these cells is complex with other cellular factors such as the levels of different G proteins and/or receptor/RAMP stoichiometry following heterologous coexpression contributing to the ultimate receptor phenotype.

Amyloid↗

PubMed1999

The pharmacokinetics and pharmaco-dynamics of Procysteine in amyotrophic lateral sclerosis.

In 13 subjects with ALS, we studied the safety and pharmacokinetic properties of Procysteine, a cysteine prodrug that increases levels of intracellular glutathione. We found that oral administration of Procysteine was safe. Procysteine enters CSF after both IV and oral dosing and accumulates to significant levels in CSF. We also observed that CSF levels of glutathione fall dramatically with aging.

Administration, Oral↗

PubMed1999

Calcitonin receptor antibodies in the identification of osteoclasts.

Osteoclasts are the cells responsible for bone resorption, and their number and rate of formation are critical in determining bone mass. To identify and quantify osteoclasts, as well as to study their formation in bone and in osteoclastogenic cultures, osteoclast-specific cell markers are required. Only the calcitonin receptor (CTR) expression unambiguously identifies osteoclasts and distinguishes them from macrophage polykaryons. However, present autoradiographic methods for CTR detection are cumbersome and time consuming. We have developed rabbit polyclonal antibodies specific for the C-terminal intracellular domain of the mouse and rat Cla CTR. These antibodies labeled HEK-293 cells stably transfected with CTR (but not untransfected HEK-293 cells). This labeling is abrogated by preabsorbing the antibodies with the recombinant antigen. The antibodies immunostained primary mouse and rat osteoclasts as well as osteoclasts in sections of mouse bone. Osteoclasts (both mononuclear and multinucleated) formed from mouse bone marrow or spleen cells cocultured with osteoblasts in the presence of 1,25 dihydroxyvitamin D3 and prostaglandin E2 were also specifically immunostained by the CTR antibodies. Cocultures incubated under conditions that did not allow osteoclastogenesis (i.e., omission of mediators or osteoblasts, or culture for less than 4 days) were not immunostained by CTR antibodies. Autoradiographic detection of 125I-labeled salmon calcitonin combined with CTR immunohistochemistry showed that both methods labeled the same cells. A CTR polyclonal antibody and monoclonal antibody F4/80 were used in combination to show immunofluorescence labeling of murine osteoclasts and macrophage populations, respectively, in marrow/osteoblast cocultures. These results indicate that simple and rapid CTR antibody-based methods can be used to identify osteoclasts, and can be used to characterize the antigenic profile of osteoclasts by using double immunofluorescence analysis.

Animals↗

PubMed1999

Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product.

Receptor activity-modifying proteins (RAMPs) are single-transmembrane proteins that transport the calcitonin receptor-like receptor (CRLR) to the cell surface. RAMP 1-transported CRLR is a calcitonin gene-related peptide (CGRP) receptor. RAMP 2- or RAMP 3-transported CRLR is an adrenomedullin receptor. The role of RAMPs beyond their interaction with CRLR, a class II G protein-coupled receptor, is unclear. In this study, we have examined the role of RAMPs in generating amylin receptor phenotypes from the calcitonin (CT) receptor gene product. Cotransfection of RAMP 1 or RAMP 3 with the human CT receptor lacking the 16-amino acid insert in intracellular domain 1 (hCTRI1-) into COS-7 cells induced specific 125I-labeled rat amylin binding. RAMP 2 or vector cotransfection did not cause significant increases in specific amylin binding. Competition-binding characterization of the RAMP-induced amylin receptors revealed two distinct phenotypes. The RAMP 1-derived amylin receptor demonstrated the highest affinity for salmon CT (IC50, 3.01 +/- 1.44 x 10(-10) M), a high to moderate affinity for rat amylin (IC50, 7.86 +/- 4.49 x 10(-9) M) and human CGRPalpha (IC50, 2.09 +/- 1.63 x 10(-8) M), and a low affinity for human CT (IC50, 4.47 +/- 0.78 x 10(-7) M). In contrast, whereas affinities for amylin and the CTs were similar for the RAMP 3-derived receptor, the efficacy of human CGRPalpha was markedly reduced (IC50, 1.12 +/- 0.45 x 10(-7) M; P <.05 versus RAMP 1). Functional cyclic AMP responses in COS-7 cells cotransfected with individual RAMPs and hCTRI1- were reflective of the phenotypes seen in competition for amylin binding. Confocal microscopic localization of c-myc-tagged RAMP 1 indicated that, when transfected alone, RAMP 1 almost exclusively was located intracellularly. Cotransfection with calcitonin receptor (CTR)I1- induced cell surface expression of RAMP 1. The results of experiments cross-linking 125I-labeled amylin to RAMP 1/hCTR-transfected cells with bis succidimidyl suberate were suggestive of a cell-surface association of RAMP 1 and the receptors. Our data suggest that in the CT family of receptors, and potentially in other class II G protein-coupled receptors, the cellular phenotype is likely to be dynamic in regard to the level and combination of both the receptor and the RAMP proteins.

Animals↗

PubMed1999

Calcitonin.

The peptide calcitonin (CT) was initially discovered in 1962 as a novel hypocalcemic hormone. This hypocalcemic response was principally due to a potent inhibitory action of CT on osteoclast mediated bone resorption and it is this action which underlies its widespread clinical use for the treatment of bone disorders, including Paget's disease, osteoporosis and hypercalcemia of malignancy. In this article we review the basic physiology of CT action, structure-function studies on CT peptides, cloning of CT receptors and the identification of isoforms of the receptor derived from alterative splicing of the receptor mRNA. We also review the state of understanding on CT receptor mediated signaling and receptor regulation, along with developing concepts of how CT peptides interact with the receptor, including how the receptors may interact with receptor activity modifying proteins to produce novel phenotypes. Finally, current therapeutic use is reviewed, and the potential for expanded use that may come with advances in delivery of peptides or CT mimetics.

Amino Acid Sequence↗

PubMed1998

Function of the rat calcitonin receptors, C1a and C1b, expressed in Xenopus oocytes.

The function of the cloned rat calcitonin receptors, C1a and C1b, was studied in Xenopus oocytes using the two-electrode voltage clamp method. In oocytes expressing the C1a receptors and the cystic fibrosis transmembrane conductance regulator (CFTR), C1a/ CFTR, application (30 sec) of either salmon calcitonin (sCT) or human calcitonin (hCT) activated currents through CFTR. In C1b/CFTR, sCT activated the currents, whereas hCT failed to elicit a response. The sCT induced currents in C1a/CFTR were similar in size to those in C1b/CFTR. Both the activation and the deactivation of sCT-induced currents were slower in C1a/ CFTR. In oocytes expressing C1a or C1b alone, application of relatively high concentrations of sCT induced small oscillatory inward currents. Application of hCT induced small inward currents in C1a alone, but failed to activate currents in C1b alone. These results demonstrate new insights into the signal transduction of calcitonin receptors.

Adenylyl Cyclases↗

PubMed1998

Characterization of binding sites for amylin, calcitonin, and CGRP in primate kidney.

Analysis of receptor distributions for 125I-labeled amylin, 125I-labeled calcitonin, and 125I-labeled calcitonin gene-related peptide (CGRP) in Macaca fascicularis kidney by in vitro autoradiography revealed distinct patterns of binding for each peptide. 125I-rat amylin bound primarily to the cortex, being associated with the distal tubule, including apparent binding to the juxtaglomerular apparatus. 125I-salmon calcitonin displayed high-density binding in the cortex with low-density binding to the medulla. Emulsion autoradiography indicated that binding was associated with both distal tubule and thick ascending limb of the loop of Henle. Intense binding was also found often over juxtaglomerular apparatus. 125I-rat CGRP-alpha exhibited low- to moderate-density binding to the inner medulla/papilla with high-density binding over small-, medium-, and large-caliber arteries. Weak binding to the glomerulus was also seen, but no binding was associated with cortical tubules. Competition binding studies, performed with each of the radioligands, revealed peptide specificity profiles for CGRP and calcitonin receptors that were similar to those described in rat. However, the monkey amylin receptors differed from those in rat, exhibiting relatively higher affinity for calcitonin peptides but reduced affinity for CGRP peptides. These studies suggest potential roles for amylin, calcitonin, and CGRP in primate renal function.

Amino Acid Sequence↗

PubMed1998

Purification of calcitonin-like peptides from rat brain and pituitary.

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.

Amino Acid Sequence↗

PubMed1997

Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

Animals↗

PubMed1997

Electrophoretic mobility and glycosylation characteristics of heterogeneously expressed calcitonin receptors.

The translated calcitonin receptor (CTR) complementary DNA sequences contain potential N-linked glycosylation sites within the extracellular N-terminus. We investigated the relative molecular mass (M(r)) and degree of N-linked glycosylation of five cloned CTRs (pig, rat C1a, rat C1b, human I1-ve, and human I1+ve), together with the pig hypothalamic CTR, to analyze the potential contribution of carbohydrate moieties to the molecular identity of these receptors. Receptors were cross-linked to 125I-salmon CT with the homobifunctional reagent bis(sulfosuccinimidyl) suberate. Autoradiographic analysis of the cross-linked receptors, following SDS-PAGE, revealed apparent M(r)S, ranging between 70,000 and 80,000 for the rat, human, and pig hypothalamic receptors. However, the cloned, expressed pig CTR was much smaller (approximately 58,000). The lower M(r) of the cloned pig CTR appeared to be due to absence of N-terminal residues, but this did not impact on ligand-receptor specificity when compared with the hypothalamic pig CTR. Cleavage under nondenaturing conditions of N-linked sugars from the CTRs using endoglycosidase F (Endo F), increased the electrophoretic mobility of all receptors, except the pig CTRs, by approximately 10 kDa. Under denaturing conditions, electrophoretic mobilities increased by approximately 30 kDa for the rat C1a, rat C1b, and humanI1-ve (expressed in human embryonic kidney-293 cells) CTRs and by approximately 20 kDa for the cloned pig, pig hypothalamic, and human CTR isoforms (expressed in baby hamster kidney cells). Competition binding studies using glycosylated and partially deglycosylated (nondenaturing conditions) receptor preparations demonstrated no significant differences in binding affinity or specificity. Thus the CTRs are N-linked glycoproteins whose degree of glycosylation is both cell-type and species dependent.

Amino Acid Sequence↗

PubMed1997

Characterization of amylin and calcitonin receptor binding in the mouse alpha-thyroid-stimulating hormone thyrotroph cell line.

Recently, a high affinity amylin binding site was identified in the mouse alpha-TSH thyrotroph cell line. In this study, we have characterized binding sites for 125I-salmon calcitonin (125I-sCT), 125I-rat alpha-calcitonin gene-related peptide (125I-CGRP), and 125I-rat amylin in alpha-TSH cells. Using 125I-CGRP or 125I-rat amylin, equilibrium was rapidly reached, and binding was fully reversible. Competition binding revealed the relative potency of peptides was sCT>amylin, CGRP>>rCT, which is similar to the specificity profile of amylin receptors characterized in rat brain. Furthermore, specific binding of 125I-rat amylin and 125I-CGRP to membrane preparations was reduced by 52% and 39%, respectively, in the presence of 20 microM GTP-gamma-s, indicating a requirement of G protein coupling for high affinity binding. In contrast, 125I-sCT binding reached equilibrium more slowly, was essentially irreversible, and was unaltered by GTP-gamma-s. Competition binding studies using 125I-sCT as radioligand demonstrated only weak interaction by CGRP or amylin, consistent with other described CT receptors. Assessment of ligand-induced cAMP accumulation and intracellular calcium signaling revealed a relative specificity profile of sCT>rCT with little or no second messenger signaling stimulated by amylin or CGRP, consistent with a C1-CT receptor phenotype. RT-PCR amplification of messenger RNA indicated that the predominant isoform was the C1a CT receptor. In cross-linking studies, 125I-rat amylin and 125I-CGRP specifically labeled a major band of relative molecular mass (Mr) approximately 80K, being approximately 10 kDa higher than the major 125I-sCT binding protein. Full deglycosylation of N-linked carbohydrates with endoglycosidase F reduced the Mr of each of the labeled proteins to approximately 50K. Cross-linked amylin or CT receptors were immunoprecipitated with C-terminally directed antimouse or antirat CT receptor antibodies but were not immunoprecipitated with nonimmune sera or antihuman CT receptor antibodies. The current data demonstrate expression of two biochemically distinct receptor phenotypes in mouse alpha-TSH cells, a CT receptor phenotype and an amylin receptor phenotype that have highly similar protein backbones.

Amino Acid Sequence↗

PubMed1996

Amylin binding in rat renal cortex, stimulation of adenylyl cyclase, and activation of plasma renin.

125I-labeled rat amylin binds to specific sites in the cortex of rat kidney, which can be distinguished from those for 125I-labeled salmon calcitonin (sCT) and 125I-labeled rat alpha-calcitonin gene-related peptide (alpha-CGRP) on the basis of regional distribution. These sites have a high affinity (approximately 1 nM) for amylin, and 125I-amylin binding is potently inhibited by the peptide antagonists AC413 and sCT-(8-32), whereas CGRP-(8-37) is a poor inhibitor of binding. Furthermore, incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) inhibits 125I-amylin binding by > 90%, indicating that binding is dependent on coupling to G proteins. In renal cortex, amylin stimulated adenylyl cyclase activity three- to fourfold, and this was inhibited by AC413 and sCT-(8-32) but not by CGRP-(8-37). Amylin activated plasma renin twofold, and this was blunted by prior administration of AC413 but not CGRP-(8-37). We speculate that amylin may play an important role in renal physiology and that in states of hyperamylinemia, as found in obesity and the insulin resistance syndrome, this peptide may be involved in the genesis and development of hypertension.

Adenylyl Cyclases↗