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Characterization of Amylin and Calcitonin Receptor Binding in the Mouse -Thyroid-Stimulating Hormone Thyrotroph Cell Line*

Neurobiology Unit (K.J.P., M.Q., M.M., G.C., P.M.S.), St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy 3065, Victoria, Australia; and Department of Medicine (D.E.M.), Royal Melbourne Hospital, Parkville 3052, Victoria, Australia

Address all correspondence and requests for reprints to: Dr. Patrick M. Sexton, Neurobiology Unit, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, 3065 Victoria, Australia. E-mail: pms{at}rubens.its.unimelb.edu.au

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, a high affinity amylin binding site was identified in the mouse -TSH thyrotroph cell line. In this study, we have characterized binding sites for ¹²⁵I-salmon calcitonin (¹²⁵I-sCT), ¹²⁵I-rat -calcitonin gene-related peptide (¹²⁵I-CGRP), and ¹²⁵I-rat amylin in -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 µM GTP--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--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 (M_r) 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 M_r 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 -TSH cells, a CT receptor phenotype and an amylin receptor phenotype that have highly similar protein backbones.

	Introduction

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AMYLIN, also known as islet amyloid polypeptide, is a hormone proposed to have a regulatory role in carbohydrate metabolism (1). Predominantly synthesized within islet ß-cells and stored within pancreatic secretory granules (2), it is cosecreted with insulin in response to glucose (3). Amylin decreases pancreatic ß-cell insulin secretion, whereas in skeletal muscle it exhibits a highly potent inhibition of insulin-stimulated incorporation of glucose into glycogen (4), in addition to the promotion of glycogen breakdown (5). More recently, specific amylin binding within rat renal cortex (6) and stimulation of PRA (6, 7) have been demonstrated, identifying a possible role for amylin in renal physiology. Furthermore, potent actions including anorexia and adipsia are induced by centrally administered amylin, suggesting a role for amylin, or its receptors in the central nervous system (8).

Composed of 37 amino acids, amylin shares approximately 50% and 18–33% sequence homology with the calcitonin gene-related peptides (CGRPs) and the calcitonins (CTs), respectively (Fig. 1). The three peptides are members of a related gene family (9) and share a number of secondary structural features including a disulfide bridged loop of 6 or 7 amino acids at the amino terminus, a C-terminally amidated aromatic residue present at the carboxy terminus, and a region of predicted amphipathic -helical structure from residues 8–18 (8–22 for salmon CT; sCT) (8).

View larger version (32K): [in this window] [in a new window]   Figure 1. Alignment of amylin sequences from different species compared with the structures of various calcitonins and calcitonin gene-related peptides.

Nonetheless, the overlap in specificity of C3-amylin receptors with the teleost CTs, along with the affinity of amylin for porcine CT receptors (16) and the strong colocalization of amylin receptors with CT receptors, has raised questions on the relationship between amylin and CT receptors (reviewed in 8 .

Biochemical characterization of amylin receptors, however, has been difficult due to a paucity of clonal cell lines with an identifiable amylin receptor phenotype. Recently, an amylin/C3-like receptor was described in the mouse -TSH thyrotroph cell line (17). In this paper, we demonstrate that both C3-amylin and C1-CT receptors are present in mouse -TSH cells and reveal that amylin binds to a receptor biochemically distinct from the CT receptor but contains a highly similar protein backbone.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hormones and chemicals
Synthetic sCT, rat -calcitonin-gene-related-peptide (CGRP), rat calcitonin (rCT), and rat amylin were obtained from Bachem (Torrance, CA). Bacitracin, forskolin, -globulins, guanosine 5'-O-3-thiotri-phosphate (GTP--s), and isobutylmethylxanthine (IBMX) were purchased from Sigma Chemical Co. (St. Louis, MO). BSA was obtained from Commonwealth Serum Laboratories Ltd. (Parkville, Australia). The cAMP antibody was a gift from Dr. P. Marley (Department of Pharmacology, University of Melbourne, Melbourne, Australia). Endoglycosidase F/N-glycosidase F (Endo F) was purchased from Boehringer Mannheim (Mannheim, Germany). The cross-linking agent bis(sulfosuccinimidyl) suberate (BS³) was obtained from Pierce Chemical Co. (Rockford, IL). The SDS-PAGE protein molecular mass standards phosphorylase b (97.4 kDa), BSA (66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), hen egg white lysozyme (14.4 kDa), and protein dye reagent were from Bio-Rad (Hercules, CA).

Iodination of peptides
Synthetic sCT was iodinated (Na¹²⁵I, Amersham, Buckinghamshire, UK) using a modification of the chloramine-T method (18). Rat--CGRP was iodinated and purified by reverse phase HPLC as previously described (18). The specific activities of ¹²⁵I-sCT and ¹²⁵I-CGRP were approximately 700 Ci/mmol and approximately 2000 Ci/mmol, respectively. ¹²⁵I-Bolton and Hunter-labeled rat amylin (¹²⁵I-rat amylin; specific activity, 2000 Ci/mmol) was obtained from Amersham.

-TSH cell culture
-TSH cells secrete only the -subunit of TSH. The stable cell line was derived from the transplantable mouse tumor MGH101 (19) and was a gift from Dr. P. Mellon (School of Medicine, University of CA, San Diego, CA). Cells were maintained in 175 cm² flasks at 37 C in a humidified atmosphere with 95%O₂/5%CO₂, in complete DMEM (Life Technologies, Grand Island, NY) containing 10% FBS (Trace Biosciences, Sydney, Australia), 80 mg/liter gentamycin (Delta West, WA, Australia), 1 mg/liter minocycline (Sigma), and 15 mM HEPES.

Cell membrane preparations
For preparation of membranes, cells were initially pelleted by centrifugation at 500 x g for 15 min at 4 C. Pellets were resuspended in 1 ml PBS (0.8% NaCl (wt/vol), 0.2% KCl (wt/vol), 8 mM NaH₂PO₄, 1.5 mM KH₂PO₄, pH 7.3; PBS) and added drop wise to 30 ml of ice-cold homogenization buffer A (1.0 mM HEPES, pH 7.4, containing 0.5 µg/ml pepstatin, 0.25 µg/ml leupeptin, 0.1 mg/ml benzamidine and 0.1 mg/ml bacitracin). The suspensions were centrifuged at 5,000 x g for 15 min at 4 C. The pellets were resuspended in 30 ml buffer A and homogenized in an Ultra-Turrax (Kinematica, Lucerne, Switzerland) at 13,000 revolutions/min for 30 sec. The homogenates were recentrifuged at 5000 x g for 15 min at 4 C. The low speed supernatants were retained and centrifuged at 100,000 x g for 1 h at 4 C. The pellets were retained and resuspended in ice-cold homogenization buffer B (50 mM HEPES, pH 7.4, containing 0.5 µg/ml pepstatin, 0.25 µg/ml leupeptin, 0.1 mg/ml benzamidine and 0.1 mg/ml bacitracin) with 3 strokes of a motorized glass-teflon homogenizer. The protein concentration was determined by the Bradford protein microassay (20) using -globulin as the protein standard. Membrane aliquots were stored at -80 C until required.

Receptor binding assays
For binding in suspension, cells were grown to confluence in 175 cm² flasks, centrifuged at 500 g and the pellets resuspended in binding buffer (DMEM containing 0.1% (wt/vol) BSA and 0.05% (wt/vol) bacitracin). Cells (2 x 10⁵) were aliquoted into 1.5 ml Eppendorf tubes (Hamburg, Germany) and incubated with the specified radioligand (100,000 cpm) in the absence (total binding), or presence of increasing concentrations of unlabeled ligand. Nonspecific binding was defined as binding in the presence of 1 µM homologous unlabeled peptide. After incubation for 1 h at 37 C, the reaction mixtures were centrifuged in a Beckman microcentrifuge for 4 min at 4 C. The pellets were overlaid with ice-cold PBS to remove unbound radioactivity and recentrifuged. The pellets were counted for radioactivity in a Packard -counter (70% efficiency) to determine bound radioactivity. The results shown are representative of at least two separate experiments performed in triplicate.

For membrane binding, assays were completed in a similar manner. Membranes were thawed, centrifuged at 12,000 x g for 10 min at 4 C, and resuspended in binding buffer (20 mM HEPES, pH 7.4, containing 5 mM MgCl₂, 5 mM KCl, 10 mM NaCl, 0.1% (wt/vol) BSA, 0.25% (wt/vol) bacitracin). Aliquots of membranes (20 µg protein for ¹²⁵I-sCT binding; 50 µg for ¹²⁵I-rat amylin or ¹²⁵I-CGRP binding) were incubated as described for suspension assays, and the results shown are representative of at least three separate experiments performed in triplicate. To examine the effect of G protein coupling on binding, additional membrane binding assays were also completed in the presence of 20 µM GTP--s. This followed initial dose-response curves with increasing concentrations of GTP--s, which established that a maximal effect of GTP--s was obtained at a concentration of 20 µM.

Dissociation constant (K_D) and receptor concentration (B_max) values for homologous radioligand-peptide competition studies were analyzed using the nonlinear iterative curve fitting program LIGAND (21). IC₅₀ values for competing peptides were derived using a four-parameter logistic fit, using the program SigmaPlot (Jandel, CA).

Time course of association
Cells were prepared as described above and incubated at the described temperature with radioligand, in either the presence or absence of 1 µM homologous unlabeled peptide. At the times indicated (t = 2, 5, 20, 60, and 120 min), samples were removed and assayed for bound radioligand as described above.

Time course of dissociation
Cells were prepared as above and incubated with radioligand in the presence or absence of 1 µM of homologous unlabeled ligand for 20 min at the specified temperature. The reaction mixtures were then centrifuged, the supernatant aspirated, and the pellets resuspended in excess (0.1 µM) unlabeled peptide, followed by further incubation at the original temperature. At the times indicated (t = 0, 2, 5, 20, 60, and 120 min), the samples were removed and assayed for bound radioligand as described above.

cAMP assay
Cells were gown to confluence in 175 cm² flasks, centrifuged at 500 x g, and resuspended in cyclase buffer (DMEM containing 0.1% (wt/vol) BSA and 1 mM IBMX). Cells (5 x 10⁵) were aliquoted into 1.5 ml Eppendorf tubes and preincubated for 20 min at 37 C. Cells were subsequently incubated for 18 min in the absence (basal) or presence of increasing concentrations of peptide. Forskolin was included to determine maximal cAMP accumulation for this system. Following incubation, the reactions were centrifuged in a Beckman microcentrifuge at 12,000 x g for 1 min at 4 C. The cells were washed with PBS and recentrifuged. The cAMP was extracted with 0.5 ml absolute ethanol. The samples were evaporated to dryness and reconstituted in buffer (50 mM sodium acetate, 1 mM theophylline). Levels of cAMP were assayed using a specific RIA (16) following acetylation of samples. Unbound radioactivity was extracted for 15 min at 4 C with 1 ml separation buffer [100 mM dipotassium hydrogen phosphate, 100 mM potassium phosphate, pH 7.4, containing 0.25% (wt/vol) BSA and 0.2% (wt/vol) charcoal[rsqb[ and separated from antibody-bound radioactivity by centrifugation for 15 min at 4,000 x g. The supernatant was aspirated, and pellets were counted on a Packard -counter. All results are representative of at least three experiments performed in triplicate.

Measurement of intracellular ionic calcium concentrations
Intracellular calcium levels ([Ca²⁺]_i) were assayed using cells in suspension culture. For experiments, the cells were centrifuged at 500 x g and washed by resuspension in 20 ml assay medium (phenol red-free DMEM and containing 1.8 mM calcium). The cells were recentrifuged and subsequently resuspended to 2 x 10⁶ cells/ml in assay medium. The autofluoresence of cells in suspension was calculated before the loading of cells with Fura-2 dye. Cells were incubated with 2 µM of Fura-2-AM (Molecular Probes, Eugene, OR) for 30 min at 37 C, 95%O₂/5%CO₂. Following loading, cells were centrifuged at 500 x g for 5 min, and washed once with 20 ml of assay medium. The supernatant was removed and the pellet resuspended in 10–20 ml assay medium at a concentration of 2 x 10⁶ cell/ml. The cells were placed in a quartz fluorescence cuvette and were kept in suspension by gentle stirring. Fluorescence (emission at 505 nm) was measured from cells excited alternatively at 340 and 380 nm using a Spex Fluorolog 2 spectrofluorometer (Spex Industries, Inc., Edison, NJ). The calibration and calculation of intracellular calcium concentrations ([Ca²⁺]_i) was performed as previously described (22). All results are representative of at least three separate experiments.

Covalent cross-linking
Chemical cross-linking was carried out on membrane preparations that were prepared as described above.

Membranes were thawed, centrifuged at 12,000 x g, and resuspended in PBS containing 0.1% (wt/vol) BSA and 0.05% (wt/vol) bacitracin. The membranes (0.6 mg of protein) were subsequently incubated for 1 h at 20 C with 4–9 nM of the specified radioligand in the presence (nonspecific binding) or absence (total binding) of 1 µM homologous unlabeled peptide. Following incubation, membranes were centrifuged in a Beckman microcentrifuge and washed once with PBS. The cross-linking reaction was initiated by the resuspension of membranes in 0.5 ml of 1 mM BS³ in PBS, and the reactions were allowed to proceed for 20 min at ice-bath temperature. Membranes were then centrifuged at 12,000 x g for 10 min and the reaction terminated by resuspension of the pellet in 0.5 ml of 0.1 M Tris/HCl, pH 7.4, containing 10 mM EDTA. Membranes were then subjected to either endoglycosidase treatment or solubilization in sample buffer (50 mM Tris/HCl, pH 6.8, containing 2% (wt/vol) SDS, 0.1% (wt/vol) bromophenol blue, 10% (vol/vol) glycerol, 100 mM DTT). Extracts were boiled for 3 min, centrifuged at 100,000 x g for 30 min at 4 C, and analyzed on 10% (vol/vol) SDS/PAGE by the method of Laemmli (23). Following electrophoresis, the gels were stained with Coomassie blue R-250, destained, dried, and exposed to phosphor screens (Molecular Dynamics, Sunnyvale, CA). The relative molecular mass (M_r) of the labeled bands was determined from a standard curve generated from the electrophoretic mobility of molecular mass markers that were coelectrophorized with the samples.

Endoglycosidase treatments
After cross-linking, labeled receptors were treated with either Endo F under reducing and denaturing conditions, or nonreducing and nondenaturing conditions. Under reducing and denaturing conditions, extracts of labeled cells were heated at 50 C for 6 min in sample buffer, diluted 1:1 to lower the SDS concentration, and then incubated overnight at 37 C with 1.25% (vol/vol) Nonidet P-40 and 2 U/ml of Endo F. Extracts were then concentrated by chloroform/methanol protein precipitation (24), pellets resuspended in sample buffer, and analyzed as described above. For treatment under nonreducing and nondenaturing conditions, cross-linked cells were resuspended in 50 mM Tris/HCl, pH 6.8, and incubated overnight at 20 C with 4 U/ml of Endo F. Membranes were then resuspended in sample buffer, boiled for 3 min, and analyzed as described above. Control samples were incubated in parallel without enzyme for all treatments.

PCR amplification of calcitonin receptor messenger RNA (mRNA)
Five micrograms of total RNA from -TSH cells was reverse transcribed using random hexamer primers and AMV reverse transcriptase (Promega, Madison, WI), for 2 h at 42 C. The resultant complementary DNAs (cDNAs) were amplified by PCR using an oligonucleotide with the sequence 5'-CCGGATAGGAGGTGGAGGATA-3' as the forward primer, and an oligonucleotide, 5'-CGGACAATGTTGAGAAG-3' as the reverse primer. The oligonucleotides are equivalent to positions 27–48 and 1564–1580, respectively, of the mouse C1b CT receptor sequence (GenBank MMU18542). A 20-µl reaction volume contained reaction buffer (60 mM Tris-HCl, 15 mM (NH₄)SO₄, 2 mM MgCl₂, pH 9.0), 0.1 mM dNTP mix, 25 pmol of each oligonucleotide, 2 µl of -TSH cDNA from the reverse transcription reaction, and 1 U of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). PCR amplification was performed in a Perkin-Elmer DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT) with 40 cycles of 1 min denaturation at 94 C, 1 min annealing at 62 C, and 2 min extension at 72 C. Aliquots of PCR reactions were analyzed by 0.8% (wt/vol) agarose gel electrophoresis containing ethidium bromide. The predicted size of PCR product for the C1a isoform of the CT receptor is approximately 1.5 kb.

Southern blot analysis
The electrophoresed agarose gel was transferred to Hybond-N membrane (Amersham) by capillary transfer in 20 x standard sodium citrate (3 M NaCl, 0.3 M sodium citrate; SSC), followed by cross-linking under UV light for 4 min. Prehybridization and hybridization were performed in hybridization buffer (15% deionized formamide, 0.6 M NaCl, 30 mM Na₂HPO₄, 18 mM NaH₂PO₄, 5 mM EDTA, 0.04% (wt/vol) Ficoll, 0.04% (wt/vol) polyvinlypyrrolidone, and 0.04% (wt/vol) BSA (Pentax Fraction V) containing 1% (wt/vol) skim milk powder and 0.01% SDS) for 2 and 16 h, respectively, at 42 C. An oligonucleotide with the sequence 5'-ACTGCGCCCGCCTGGAATC-3', homologous to positions 101–119 in the mouse C1b CT receptor cDNA sequence, was ³²P--ATP end labeled using 10 U polynucleotide kinase (Boehringer Mannheim) and used as a probe for mouse CT receptor cDNA. The hybridized filter was washed with SSC and SDS for 15 min at 42 C with increasing stringency, ranging from 2 x SSC, 0.1% SDS (wt/vol) to 0.1 x SSC, 0.1% SDS, followed by exposure to a phosphor screen for 16 h. Subsequently, the hybridized filter was stripped of radionucleotide by boiling in 0.1 x SSC, 0.1% SDS solution and hybridized to a ³²P--ATP end labeled oligonucleotide (5'-CATCATAACACACATATGTGGACAATGCAGTG-3') specific for the insert sequence of the C1b isoform of the CT receptor (position 1168–1199 of the mouse CT receptor sequence).

Generation of anti-CT receptor antibodies
To produce maltose binding protein-CT receptor fusion proteins, oligonucleotides were designed to PCR amplify DNA fragments encoding the C-terminal amino acids of human C1a (406–490), rat C1a (390–487), and mouse C1b (427–515) CT receptors. The forward primer used to PCR amplify human, mouse, and rat DNA was 5'-AATTCGAATTCATCTACTGCTTCTGCAAC-3' containing tandem EcoRI sites on the 5' end. The reverse primer used on rat and mouse template was 5'-GTCGACGTCGACCATTCAAGCGGATG-3', whereas the human antisense primer sequence was 5'-GTCGACGTCGACTTCAAGCAGATGACTCTTG-3' with both reverse primers containing tandem SalI sites incorporated into the 3' end. The mouse cDNA was generated from 5 µg of total RNA from -TSH cells and was reverse transcribed using random hexamer primers and AMV reverse transcriptase for 2 h at 42 C. The 20-µl PCR reactions contained 60 mM Tris-HCl, 15 mM (NH₄)SO₄, 2 mM MgCl₂, pH 9.0, -TSH cDNA (2 µl), 25 pmol of each primer, 0.1 mM dNTP, and 1.0 U Taq polymerase (Perkin Elmer). PCR amplification of the human and rat C-terminal CT receptor was performed as described above with the exception of DNA template. One hundred nanograms of recombinant DNA cloned in the vectors Zem 228cc (25) and pcDNA₁neo (26) were used as the respective human and rat DNA templates. PCR reactions underwent an initial denaturation at 95 C for 5 min, followed by 35 cycles of denaturation for 30 sec at 95 C, annealing for 30 sec at 60 C, extension for 1 min at 72 C, with a final elongation step for 10 min at 72 C. PCR reactions were electrophoresed on a 1.0% (wt/vol) agarose gel and transferred to Hybond-N nylon filters for Southern blot analysis as described above. Transferred PCR products were probed with a conserved internal C-terminal CT receptor oligonucleotide (5'-TGTAACCATGAGGTGCAA -3') and hybridized filters were washed to 1.0 x SSC, 0.1% SDS stringency, before exposure to a phophor screen for 16 h. Once the identity of PCR fragments was confirmed, samples were electrophoresed and purified from a 1.0% low melting point agarose gel using the QIAEX II gel extraction kit (QIAGEN, Hilden, Germany).

A fusion protein of the CT receptor fragment with Escherichia coli maltose binding protein (MBP) was constructed using a protein fusion and purification system according to the manufacturer’s instruction (New England Biolabs, Beverly, MA). C-terminal CT receptor DNA was directionally cloned into the 3' end of the MBP gene in the pMAL-c2 expression vector. Purified PCR amplified DNA fragments were subcloned into the pGEM-T expression vector (Promega) and excised by double digestion with EcoRI (New England Biolabs) and SalI (Promega). DNA fragments were subsequently ligated into an EcoRI and SalI digested pMAL-c2 vector and transformed into electrocompetent TB1 Escherichia coli cells. Clones were confirmed by dideoxy nucleotide chain termination sequencing (27). Fusion proteins were subsequently expressed, and purified by affinity chromatography using amylose resin (New England Biolabs).

Rabbits were injected sc with either 1.0 mg or 0.5 mg of fusion protein emulsified with Freund’s complete adjuvant. Subsequent injections were at one month intervals with 0.2 mg of fusion protein mixed with Freund’s incomplete adjuvant. Ten-microliter bleeds were taken 14 days after after immunizations. Antibody titers were assayed by Westen blot with both fusion protein, and membrane preparations of cells expressing CT receptors. Receptor naïve cells were used as a control.

Western blot analysis of -TSH cell membranes
Membrane preparations were thawed, microfuged for 5 min, and resuspended in electrophoresis sample buffer. Samples were heated at 55 C for 6 min before loading approximately 10 µg of total protein onto a 10% (vol/vol) SDS-PAGE gel as described above. Following electrophoresis, gels were transferred to nitrocellulose filters (0.2 µm) and blocked overnight in blocking solution (50 mM Tris base, 150 mM NaCl, pH 7.5, and 1% Tween 20; Tris-buffered saline:Tween (TBST), containing 4% (wt/vol) skim milk powder). The filters were incubated with antisera (1:200 or 1:500) for 1 h, washed twice with TBST and twice with blocking solution, followed by a 30-min incubation with a 1:10³ dilution of sheep antirabbit IgG coupled to horseradish peroxidase (Boehringer Mannheim). Membranes were washed four times in TBST and exposed to ECL Western blotting detection reagents (Amersham). Immunoreactive proteins were visualized by exposure to x-ray film.

Immunoprecipitation of cross-linked receptor proteins
-TSH membrane proteins were cross-linked to either ¹²⁵I-sCT or ¹²⁵I-rat amylin as described above. The membranes were subsequently centrifuged and pellets resuspended in 75 µl of SUTE (1% (wt/vol) SDS, 8 M Urea, 10 mM Tris, pH 7.5, 10 mM EDTA) and warmed to 45 C for 10 min. Four hundred microliters of immunoprecipitation buffer (15 mM NaH₂PO₄, pH 7.4, 150 mM NaCl, 2% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.5% (wt/vol) deoxycholate; IPB), containing 0.4 mM PMSF and 0.05 mM leupeptin was added, followed by the addition of 8 µl of either nonimmune sera or antimouse, antirat, or antihuman CT receptor antisera. Membranes were subsequently incubated overnight on a rotating wheel at 4 C. Following primary incubation membranes were centrifuged at 12,000 x g for 15 min, and the supernatants transferred into siliconized 1.5-ml Eppendorf tubes containing 15 µl of Protein A Sepharose beads (Pharmacia Biotech, Uppasala, Sweden) that had been preincubated in Tris-buffered saline (10 mM Tris, pH 7.5, 50 mM NaCl; TBS) containing 0.5% BSA. Samples were incubated on a rocking platform for 2.5 h at room temperature and centrifuged briefly to pellet beads. The supernatant was aspirated and beads washed twice with 0.5 ml of IPB, containing 0.5% BSA, followed by a 25 min wash with 10 ml of the same. Beads were pelleted by centrifugation and subsequently washed for 20 min in 10 ml TBS containing 0.5% BSA following aspiration of the supernatant. Beads were finally washed twice in 0.5 ml of TBS, the buffer aspirated, the beads overlayed with SDS sample buffer, and warmed to 48 C for 8 min. Beads were pelleted by centrifugation and the supernatant loaded on a 10% SDS-PAGE gel. Following electrophoresis, gels were stained with Coomassie blue R-250, destained, dried and exposed to phosphor screen for 3 days.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Binding kinetics
The rate of sCT, CGRP, and rat amylin association to and dissociation from -TSH binding sites was examined at 4 C, 22 C, and 37 C in cell suspension. Ligand binding increased over time, with ¹²⁵I-CGRP (Fig. 2B) and ¹²⁵I-amylin (Fig. 2C) displaying similar kinetics, both exhibiting rapid association to binding sites which reached equilibrium by 20 min. Binding of ¹²⁵I-sCT (Fig. 2A) was slower, reaching only 80% of maximum binding at 20 min.

View larger version (14K): [in this window] [in a new window]   Figure 2. Association of 125I-sCT (A), 125I-CGRP (B), or 125I-amylin (C) binding to -TSH cells in suspension. • 4 C, 22 C, 37 C. Values are mean ± SEM of triplicate determinations from a representative experiment performed at least three times.

View larger version (13K): [in this window] [in a new window]   Figure 3. Dissociation of 125I-sCT (A), 125I-CGRP (B) or 125I-amylin (C) binding from -TSH cells in suspension. • 4 C, 22 C, 37 C. Cells were incubated with radioligand in the presence (open symbols) or absence (closed symbols) of 10-6 M unlabeled homologous peptide, for 20 min at the indicated temperature. Unbound radiolabel was removed and the cells further incubated at the original temperature in the presence of excess (10-7 M) unlabeled peptide. At the times indicated, cells were assayed for bound radioligand. Values are mean ± SEM of triplicate determinations from a representative experiment performed at least twice.

View larger version (16K): [in this window] [in a new window]   Figure 4. Competition of 125I-sCT (A), 125I-CGRP (B), or 125I-amylin (C) binding to -TSH cell suspensions at 37 C. • sCT, rat amylin, rat CT, -rat CGRP. Values are mean ± SEM of triplicate determinations from a representative experiment performed at least three times.

View this table: [in this window] [in a new window]   Table 1. IC50 values for sCT, rat CGRP, and rat amylin in competition for 125I-sCT 125I-rat CGRP or 125I-rat amylin binding to -TSH receptors

View larger version (16K): [in this window] [in a new window]   Figure 5. Competition of 125I-sCT (A), 125I-CGRP (B), or 125I-amylin (C) binding to -TSH cell membranes at 37 C. • sCT, rat amylin, rat CT, -rat CGRP. Values are mean ± SEM of triplicate determinations from a representative experiment performed at least three times.

GTP--s

Competition binding studies in the presence or absence of GTP--s demonstrated that the decrease in binding observed with ¹²⁵I-rat amylin or ¹²⁵I-CGRP was due to a reduction in the number of binding sites, with no significant change in K_D values for homologous peptides (Table 2). The IC₅₀ values for nonhomologous competing peptides were also essentially unchanged, with the exception of amylin in competition for ¹²⁵I-CGRP binding (Table 3). In this system, the affinity of amylin was reduced approximately 10-fold in the presence of GTP--s, revealing a specificity profile similar to that demonstrated in whole cell suspension assays (Table 3 vs. Table 1).

View this table: [in this window] [in a new window]   Table 2. Effect of GTP--s on ligand affinity and receptor concentration in membrane binding assays

View this table: [in this window] [in a new window]   Table 3. Effect of GTP--s on peptide affinities in membrane binding assays

Second messenger responses
Calcitonin receptors, in addition to other members of the CT/secretin subfamily of seven transmembrane domain G protein coupled receptors, demonstrate heterogeneity in G protein coupling and modulate multiple second messengers inducing rises in cAMP levels and mobilization of intracellular calcium.

Receptor-mediated induction of cAMP by sCT, rCT, amylin, and CGRP was studied in cell suspension. The rel-ative potencies of ligands in increasing cAMP was sCT>rCT>>amylin, CGRP, with induction of cAMP accumulation by amylin and CGRP occurring only at high ligand concentrations (10–100 nM) (Fig. 6).

View larger version (23K): [in this window] [in a new window]   Figure 6. Ligand-induced cAMP accumulation in -TSH cell suspensions at 37 C. • sCT, rat amylin, rat CT, -rat CGRP, forskolin. Data are expressed as fold-increase above basal levels. Basal levels ranged from 2–8 pmol/ml. The maximal response with 10-4 M forskolin was approximately 10- to 40-fold above basal levels. Values are mean ± SEM of triplicate determinations from a representative experiment performed four times.

View larger version (15K): [in this window] [in a new window]   Figure 7. Salmon CT (A), rat CT (B), amylin (C), or CGRP (C)-induced mobilization of intracellular calcium measured by the Fura-2 method (22). The figure shows individual traces of experiments performed 3 or 4 times for each peptide concentration. In each experiment, after responses had reached plateau, cells were restimulated with 10-7 M sCT to monitor desensitization of the CT response.

View larger version (47K): [in this window] [in a new window]   Figure 8. Autoradiography of 125I-sCT (A), 125I-amylin (B), or 125I-CGRP (C) cross-linked to -TSH cell membrane receptors using BS3. Cross-linked samples were analyzed by SDS-PAGE and radiolabeled proteins visualized by exposing dried gels to phosphor screens. Samples were incubated overnight in the presence (T+) or absence (T) of Endo F under nonreducing and nondenaturing conditions (left panels), or reducing and denaturing conditions (right panels). Lanes N show binding in the presence of 1 µM of unlabeled homologous peptide. The site of migration of molecular weight markers is indicated on the left side of each figure. The data shown is from an individual experiment. Pooled data are displayed in Table 4.

View this table: [in this window] [in a new window]   Table 4. Relative Mr of -TSH receptors cross-linked to radiolabeled sCT, rat CGRP, or rat amylin in the presence or absence of endoglycosidase (Endo F) under nonreducing and nondenaturing conditions, or reducing and denaturing conditions

Immunoprecipitation of cross-linked receptor
Immunoprecipitation studies were performed using antibodies raised against the C-terminus of CT receptors from mouse, rat or human origin. Both antimouse and antirat CT receptor antibodies precipitated either ¹²⁵I-sCT or ¹²⁵I-rat amylin cross-linked receptor proteins (Fig. 9B). However, there was little or no recognition of receptor proteins by nonimmune sera or antihuman CT receptor antibodies (Fig. 9B).

View larger version (30K): [in this window] [in a new window]   Figure 9. Immunoprecipitation of amylin and CT binding proteins from -TSH cells with anti-CT receptor antibodies. A, Autoradiography of 125I-sCT and 125I-rat amylin (125I-rAm) cross-linked to -TSH membranes. Samples were incubated in the absence (total binding) or presence (nonspecific binding) of 1 µM unlabeled homologous peptide. B, Autoradiography of cross-linked membranes (from A) immunoprecipitated with antibodies raised against the C-terminus of the mouse CT receptor (anti-mCTR), the rat CT receptor (anti-rCTR), the human CT receptor (anti-hCTR) or nonimmune sera. The relative molecular mass of the cross-linked proteins is indicated on the left of panel A (x1000). The data shown is from an individual experiment (n 3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In radioligand cross-linking studies, ¹²⁵I-sCT bound predominantly to a protein of M_r approximately 70K, with low level binding to a M_r approximately 80K protein, which presumably represents binding to the C3-amylin receptor protein. The approximately 70-kDa protein was insensitive to endoglycosidase treatment under aqueous conditions. This contrasted with the amylin receptor protein where approximately 10 kDa of carbohydrate was cleaved under aqueous conditions. Both the amylin and CT receptor proteins contained approximately 20 kDa of carbohydrate, which required strong denaturation of the protein for cleavage.

Despite the difference in aqueous-accessible carbohydrate between the receptors, it is unclear whether this would contribute to the observed receptor phenotype. Cloned, heterogeneously expressed rat C1a or hCTR_I1-receptor both contain approximately 10 kDa of aqueous accessible carbohydrate, and its removal has no impact on receptor affinity or specificity (20). This observation is consistent with the aqueous accessible carbohydrate being directed away from the binding pocket of the protein. Similarly, the pig CT receptor, which has relatively high affinity for amylin, contained only approximately 20 kDa of aqueous inaccessible carbohydrate, similar to the major -TSH C1-CT binding protein (20). In other cell lines, M_rs of approximately 70K or approximately 80K have been observed for CT receptors expressing a C1 phenotype (29, 33, 34, 35, 36), suggesting that the level of glycosylation is not a critical factor in defining receptor phenotype.

It was previously suggested, on the basis of poor competition of binding with rat CT, that only the C3-amylin receptor phenotype exists in -TSH cells (19); however, our data clearly supports the existence of at least two distinct receptors; a C1-type CT receptor in addition to the C3-amylin receptor. The specificity of peptides in stimulating rises in cAMP and intracellular calcium were reflective of isolated, cloned C1 CT receptors (30), and this is supported by RT-PCR which demonstrated production of C1a CT receptor mRNA. Moreover, the potency of rCT, in second messenger stimulation, at the -TSH cells is similar to that of rCT at other rodent CT receptors (30, 37, 38), indicating, in contrast to the conclusions of Hannah and colleagues (17), that rCT is a viable physiological ligand for action in the thyrotroph cells. The poor efficacy of rCT in competition for ¹²⁵I-sCT binding may be due to relatively low level of G protein (as evidenced by the weak forskolin response) in this cell line.

There was no apparent [Ca²⁺]_i mobilization and only a weak cAMP induction in response to administered amylin, indicating that, at least for -TSH cells, the C3-amylin receptor does not couple to either G_s or G_q mediated signaling pathways. Nonetheless, binding of amylin was inhibited in the presence of GTP--s, consistent with G protein-mediated signaling. In L6 myocytes, amylin is reported to act via a cAMP-independent pathway (39), whereas in Rin m5F cells decreases cAMP through pertusis toxin sensitive G_i proteins (40). As such, activation of G_i may underlie the lack of amylin cAMP responsiveness observed in the current study.

Analysis of competition binding studies indicated similar relative potencies for whole cell and membrane preparations. In exception to this was the efficacy of amylin in competition for ¹²⁵I-CGRP binding, where amylin was 10-fold less potent in whole cells. This discrepancy is likely due to the decreased G protein coupling in whole cells (where GTP is generally in excess), and this is supported by the specific 10-fold increase in amylin IC₅₀ in membrane preparations treated with GTP--s. Uncoupling of receptor G protein interactions with GTP--s had greatest impact on ¹²⁵I-amylin binding, with a 44% loss of high affinity binding sites, whereas ¹²⁵I-CGRP binding sites decreased by 38%. This difference in G protein-dependent binding presumably underlies the greater impact of GTP--s on amylin competition for ¹²⁵I-CGRP binding. In contrast to binding of amylin or CGRP, GTP--s had minimal impact on binding of ¹²⁵I-sCT to -TSH membranes. This lack of effect, of G protein uncoupling on sCT binding, is likely to reflect both a relatively high affinity of sCT for inactive state receptor and the essentially irreversible nature of sCT binding to active state receptor (Hilton, J. M., M. Dowton, S. Houssami, and P. M. Sexton, manuscript submitted).

While very little data are available on murine amylin receptors, the specificity of peptides in competition for ¹²⁵I-amylin or ¹²⁵I-CGRP to -TSH cells, with sCT CGRP amylin > rCT, is similar to the C3-amylin binding sites described in rat accumbens nucleus and kidney (6, 13, 14), although the relative efficacy of amylin is slightly weaker in -TSH cells than in rat tissue. As described above, this may, in part, be a reflection of the conditions of assay. In the rat, a similar specificity profile is also seen for physiological responses in skeletal muscle (15), indicating that C3-amylin receptors mediate the metabolic actions of the hormone.

Evidence for the existence of CT or C3-amylin receptors in intact anterior pituitary is limited. Recent studies have failed to detect either CT or amylin binding to the anterior pituitary (14, 41); however, this may be due to the relatively low abundance of thyrotroph cells. Thus, it is possible that the "spots" of high level ¹²⁵I-sCT binding in the anterior pituitary, seen in an early study of pituitary CT receptors (42), may represent clusters of thyrotroph cells in the pituitaries of these animals. There is accumulating evidence for the existence of endogenous sCT-like peptides within the anterior pituitary, and these are hypothesized to be physiological regulators of anterior pituitary function, in particular of PRL secretion (43, 44). Similarly, a sCT-like peptide is secreted from the -TSH cells used in the present study (17). As sCT has high affinity for both C1 and C3 receptors, the endogenous pituitary CT peptides may provide a potential autocrine regulatory system for the thyrotroph cell, acting via the receptors described in the current investigation. However, as we were unable to determine the mechanism of C3-receptor-mediated signaling, it is unclear what the physiological significance of having both C1 and C3 receptor phenotypes is.

The molecular identity of C3-amylin receptors remains unclear. This study provides direct evidence that amylin receptors may exhibit distinct biochemical, as well as pharmacological, differences from classical C1-type CT receptors. Nonetheless, the cross-recognition of amylin and CT receptors by anti-CT receptor antibodies, raised against the hypervariable C-terminus of the receptor, provides strong evidence to suggest that both receptors are the product of the same gene.

	Footnotes

 
¹ See http://swift.embl-heidelberg.de/7tm.

Received January 27, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Cooper GJS 1994 Amylin compared with calcitonin gene-related peptide: structure, biology, and relevance to metabolic disease. Endocr Rev 15:163–201[CrossRef][Medline]
2. Lukinius A, Wilander E, Westermark GT, Engstrom U, Westermark P 1989 Co-localisation of islet amyloid polypeptide and insulin in the ß cell secretory granules of the human pancreatic islets. Diabetologia 32:240–244[CrossRef][Medline]
3. Bretherton-Watt D, Ghatei MA, Jamal H, Gilbey SG, Jones PM, Bloom SR 1992 The physiology of calcitonin gene-related peptide in the islet compared with that of islet amyloid polypeptide (amylin). Ann NY Acad Sci 657:299–312[Medline]
4. Pittner RA, Albrandt K, Beaumont K, Gaeta LSL, Koda JE, Moore CX, Rittenhouse J, Rink TJ 1994 Molecular physiology of amylin. J Cell Biochem 55S:19–28
5. Young AA, Mott DM, Stone K, Cooper GJS 1991 Amylin activates glycogen phosphorylase in the isolated soleus muscle of the rat. FEBS Lett 281:149–151[CrossRef][Medline]
6. Wookey PJ, Tikellis C, Du H-C, Qin H-F, Sexton PM, Cooper ME 1996 Amylin binding in rat renal cortex, stimulation of adenyl cyclase, and activation of plasma renin. Am J Physiol 39:F289–F294
7. Cooper ME, McNally PG, Phillips PA, Johnston CI 1995 Amylin stimulates plasma renin concentration in humans. Hypertension 26:460–464[Abstract/Free Full Text]
8. Sexton PM, Perry KJ 1996 Amylin receptors in the central nervous system. Recent Results Dev Neurochem 1:157–166
9. Born W, Fischer JA 1993 Calcitonin gene products: molecular biology, chemistry, and actions. In: Mundy GR, Martin TJ (eds) Handbook of Experimental Pharmacology. Physiology and Pharmacology of bone. Berlin Heidelberg:Springer-Verlag, pp 569–616
10. Alam ASMT, Moonga BS, Bevis PJR, Huang CLH, Zaidi M 1993 Amylin inhibits bone resorption by a direct effect on the motility of rat osteoclasts. Exp Physiol 78:183–196[Abstract]
11. Young AA, Rink TJ, Wang MW 1993 Dose-response characteristics for the hyperglycemic, hyperlactemic, hypotensive and hypocalcemic actions of amylin and calcitonin gene-related peptide-I (CGRP) in the fasted anaesthetized rat. Life Sci 52:1717–1726[CrossRef][Medline]
12. Brain SD, Wimalawansa SJ, MacIntyre I, Williams TJ 1990 The demonstration of vasodilator activity of pancreatic amylin amide in the rabbit. Am J Pathol 136:487–490[Abstract]
13. Beaumont K, Kenney MA, Young AA, Rink TJ 1993 High affinity amylin binding sites in rat brain. Mol Pharmacol 44:493–497[Abstract]
14. Sexton PM, Paxinos G, Kenney MA, Wookey PJ, Beaumont K 1994 In vitro autoradiographic localization of amylin binding sites in rat brain. Neuroscience 62:553–567[CrossRef][Medline]
15. Beaumont K, Moore CX, Pittner RA, Prickett KS, Gaeta LSL, Rink TJ, Young AA 1995 Differential antagonism of amylin’s metabolic and vascular actions with amylin receptor antagonists. Can J Physiol Pharmacol 73:1025–1029[Medline]
16. Sexton PM, Houssami S, Brady CL, Myers DE, Findlay DM 1994 Amylin is an agonist of the renal porcine calcitonin receptor. Endocrinology 134:2103–2107[Abstract]
17. Hanna FWF, Smith DM, Johnstone CF, Akinsanya KO, Jackson ML, Morgan, DGA, Bhogal R, Buchanan KD, Bloom, SR 1995 Expression of a novel receptor for the calcitonin peptide family and a salmon calcitonin-like peptide in the -thyrotropin thyrotroph cell line. Endocrinology 136:2377–2382[Abstract]
18. Sexton PM, McKenzie JS, Mendelsohn FAO 1988 Evidence for a new subclass of calcitonin/calcitonin gene-related peptide binding site in rat brain. Neurochem Int 12:323–335[CrossRef]
19. Akerblom IA, Ridgway EC, Mellon PL 1990 An -subunit-secreting cell line derived from a mouse thyrotrope tumor. Mol Endocrinol 4:589–596[CrossRef][Medline]
20. Quiza M, Dowton M, Perry KJ, Sexton PM 1997 Electrophoretic mobility and glycosylation characteristics of heterogeneously expressed calcitonin receptors. Endocrinology 138:530–539[Abstract/Free Full Text]
21. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220–239[CrossRef][Medline]
22. Williams DA, Fay FS 1990 Intracellular calibration of the fluorescent calcium indicator Fura-2. Cell Calcium 11:75–83[CrossRef][Medline]
23. Laemmli UK 1970 Cleavage of proteins during assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
24. Wessel D, Flügge UI 1984 A method for the quantitative recovery of proteins in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143[CrossRef][Medline]
25. Kuestner RE, Elrod R, Grant FJ, Hagen FS, Kuijper JL, Matthewes SL, O’Hara PJ, Sheppard PO, Stroop SD, Thompson DL, Whitmore TE, Findlay DM, Houssami S, Sexton PM, Moore EE 1994 Cloning and characterization of an abundant subtype of the human calcitonin receptor. Mol Pharmacol 46:246–255[Abstract]
26. Sexton PM, Houssami S, Hilton JM, O’Keeffe LM, Center RJ, Gillespie MT, Darcy P, Findlay DM 1993 Identification of brain isoforms of the rat calcitonin receptor. Mol Endocrinol 7:815–821[Abstract/Free Full Text]
27. Sanger F, Nicklen S, Coulson AR 1977 DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci 74:5463–5467[Abstract/Free Full Text]
28. Fischer JA, Sagar SM, Martin JB 1981 Characterization and regional distribution of calcitonin binding sites in the rat brain. Life Sci 29:663–671[CrossRef][Medline]
29. Nicholson GC, D’Santos CS, Evans T, Moseley JM, Kemp BE, Michalangeli VP, Martin TJ 1988 Human placental calcitonin receptors. Biochem J 250:877–882[Medline]
30. Houssami S, Findlay DM, Brady CL, Myers DE, Martin TJ, Sexton PM 1994 Isoforms of the rat calcitonin receptor: consequences for ligand binding and signal transduction. Endocrinology 135:183–190[Abstract]
31. Moore EE, Kuestner RE, Stroop SD, Grant FJ, Matthewes SL, Brady CL, Sexton PM, Findlay DM 1995 Functionally different isoforms of the human calcitonin receptor result from alternative splicing of the gene transcript. Mol Endocrinol 9:959–968[Abstract]
32. Chang C-P, Pearse RV-II, O’Connell S, Rosenfeld MG 1993 Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187–1195[CrossRef][Medline]
33. Sexton PM, Schneider HG, D’Santos CS, Mendelsohn FAO, Kemp BE, Moseley JM, Martin TJ, Findlay DM 1991 Reversible calcitonin binding to solubilized sheep brain binding sites. Biochem J 273:179–184
34. D’Santos CS, Nicholson GC, Moseley JM, Evans T, Martin TJ, Kemp BE 1988 Biologically active, derivatizable salmon calcitonin analog: design, synthesis, and applications. Endocrinology 123:1483–1488[Abstract]
35. Moselely JM, Smith P, Martin TJ 1986 Identification of the calcitonin receptor by chemical cross-linking and photoaffinity labelling in human cancer cell lines. J Bone Miner Res 1:293–297[Medline]
36. Moseley JM, Findlay DM, Martin TJ, Gorman JJ 1982 Covalent cross-linking of a photoactive derivative of calcitonin to human breast cancer cell receptors. J Biol Chem 257:5846–5851[Abstract/Free Full Text]
37. Houssami S, Findlay DM, O’Keefe LM, Martin TJ, Sexton PM 1994 Heterogeneity in ligand recognition of calcitonin receptors. Endocr J (UK) 2:127–134
38. Chao W-TH, Forte LR 1982 Rat kidney cells in primary culture: hormone-mediated densensitization of the adenosine 3', 5'-monophosphate response to parathyroid hormone and calcitonin. Endocrinology 111:252–259[Abstract]
39. Kreutter DK, Orena SJ, Torchia AJ Contillo LG, Andrews GC, Stevenson RW 1993 Amylin and CGRP induce insulin resistance via a receptor distinct from cAMP-coupled CGRP receptor. Am J Physiol 264:E606–E613
40. Suzuki S, Murakami M, Abe S, Satoh Y, Shintani S, Ishizuka J, Suzuki K, Thompson JC, Toyota T 1992 The effects of amylin on insulin secretion from Rin m5F cells and glycogen synthesis and lipogenesis in rat primary cultured hepatocytes. Diabetes Res Clin Pract 15:77–84[CrossRef][Medline]
41. Hilton JM, Chai SY, Sexton PM 1995 In vitro autoradiographic localisation of the calcitonin receptor isoforms, C1a and C1b, in rat brain. Neuroscience 69:1223–1237[CrossRef][Medline]
42. Maurer R, Marbach P, Mousson R 1983 Salmon calcitonin binding sites in rat pituitary. Brain Res 261:346–348[CrossRef][Medline]
43. Shah GV, Deftos LJ, Crowley WR 1993 Synthesis and release of calcitonin-like immunoreactivity by anterior pituitary cells: evidence for a role in paracrine regulation of prolactin secretion. Endocrinology 132:1367–1372[Abstract]
44. Shah GV, Pedchenko V, Stanley S, Li Z, Samson WK 1996 Calcitonin is a physiological inhibitor of prolactin secretion in ovariectomized female rats. Endocrinology 137:1814–1822[Abstract]

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