Investigation of a novel binding site for the calcitonin gene related peptide (CGRP) family of peptides in rat nucleus accumbens

High affinity amylin binding sites are present in the rat nucleus accumbens. These sites bind [125I]amylin with an affinity of 27 pM and have high affinity for salmon calcitonin (sCT) and moderately high affinity for calcitonin gene related peptide (CGRP). N-terminally truncated peptides were tested for their ability to compete for [125I]amylin binding to these sites and to antagonize the metabolic and vascular actions of amylin. CGRP(8–37), sCT(8–32), and ac-[Asn30,Tyr32]sCT(8–32) (AC187) inhibited [125I]amylin binding to rat nucleus accumbens. Order of potency at inhibiting amylin binding (AC187 > sCT(8–32) > CGRP(8–37)) differed from the order of potency at inhibiting [125I]CGRP binding to SK-N-MC neuroblastoma cells (CGRP(8–37) > AC187 > sCT(8–32)). AC187 was the most potent antagonist of amylin's effects on isolated rat soleus muscle glycogen metabolism, and it was more effective than either sCT(8–32) or CGRP(8–37) at reducing amylin-stimulated hyperlactemia in rats. In contrast, CGRP(8–37) was the most potent peptide at antagonizing amylin-induced hypotension in rats. Amylin's hypotensive actions appear to be mediated by a weak action at CGRP receptors, while its metabolic actions are mediated by receptors with a distinct antagonist profile. AC187 is a potent antagonist of amylin binding sites in nucleus accumbens and of amylin's metabolic actions.Key words: amylin, calcitonin gene related peptide, diabetes, skeletal muscle, peptide receptors.

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Solubilization and Characterization of Calcitonin Gene-Related Peptide Binding Site from Porcine Spinal Cord

Journal of Neurochemistry ◽

10.1111/j.1471-4159.1988.tb02936.x ◽

1988 ◽

Vol 50 (2) ◽

pp. 480-485 ◽

Cited By ~ 15

Author(s):

Osamu Hiroshima ◽

Yoshihisa Sano ◽

Teruaki Yuzuriha ◽

Chiyuki Yamato ◽

Akira Saito ◽

...

Keyword(s):

Spinal Cord ◽

Binding Site ◽

Peptide Binding ◽

Calcitonin Gene Related Peptide ◽

Related Peptide ◽

Calcitonin Gene ◽

Peptide Binding Site

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Influences of calcitonin gene-related peptide on mu opioid receptors in nucleus accumbens neurons of rats

Neuropeptides ◽

10.1016/j.npep.2012.10.008 ◽

2013 ◽

Vol 47 (2) ◽

pp. 125-131 ◽

Cited By ~ 5

Author(s):

Hailiang Yan ◽

Long-Chuan Yu

Keyword(s):

Nucleus Accumbens ◽

Opioid Receptors ◽

Calcitonin Gene Related Peptide ◽

Mu Opioid Receptors ◽

Mu Opioid ◽

Related Peptide ◽

Calcitonin Gene

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A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors

Biochemical Journal ◽

10.1042/bj3180241 ◽

1996 ◽

Vol 318 (1) ◽

pp. 241-245 ◽

Cited By ~ 36

Author(s):

Hedley A COPPOCK ◽

Ali A OWJI ◽

Stephen R BLOOM ◽

David M SMITH

Keyword(s):

Protein Kinase ◽

Adenylate Cyclase ◽

Binding Site ◽

Binding Sites ◽

Calcitonin Gene Related Peptide ◽

Dependent Protein Kinase ◽

Related Peptide ◽

Camp Dependent Protein Kinase ◽

Calcitonin Gene ◽

Dependent Protein

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22±0.04 nM (mean±S.E.M.) and a concentration of binding sites (Bmax) of 0.95±0.19 pmol/mg of protein (mean±S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8–37) competed weakly at this site (IC50 > 10 and 601±298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13±0.01 nM; Bmax = 0.83±0.10 pmol/mg of protein) where both CGRP(8–37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15±0.12 and 8.68±0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site–ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8–37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

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Cross-reactivity of amylin with calcitonin-gene-related peptide binding sites in rat liver and skeletal muscle membranes

Biochemical Journal ◽

10.1042/bj2770139 ◽

1991 ◽

Vol 277 (1) ◽

pp. 139-143 ◽

Cited By ~ 45

Author(s):

A Chantry ◽

B Leighton ◽

A J Day

Keyword(s):

Skeletal Muscle ◽

Binding Site ◽

Rat Liver ◽

Specific Binding ◽

Calcitonin Gene Related Peptide ◽

Cross Reactivity ◽

Scatchard Analysis ◽

Human Calcitonin ◽

Related Peptide ◽

Calcitonin Gene

This study examines whether the high degree of sequence identity between amylin and calcitonin-gene-related peptide (CGRP) is reflected in their cross-reactivity at the level of membrane receptor binding. Rat liver plasma membranes contain a specific saturable binding site for 125I-labelled human CGRP-1. Binding reached equilibrium within 30 min and was rapidly reversed by re-incubating membranes in the presence of 1 microM human CGRP. In addition, the presence of 50 mM- or 500 mM-NaCl lowered specific binding by 30% and 77% respectively. Scatchard analysis was consistent with a single high-affinity site with a dissociation constant (Kd) of 0.125 nM and binding capacity (Bmax.) of 580 fmol/mg of membrane protein. Specific binding of 125I-labelled human CGRP-1 to both liver and skeletal muscle membranes was inhibited by human CGRP-1 [IC50 (concn. causing half-maximal inhibition of binding) 0.1-0.3 nM], and rat amylin (IC50 10 nM), but not by human calcitonin. Covalent cross-linking of 125I-CGRP to its binding site in rat skeletal muscle and liver membranes resulted in labelling of a major species of about 70 kDa under reducing conditions and about 55 kDa under alkylating conditions, as visualized on SDS/PAGE. These radiolabelled species were absent in the presence of CGRP or amylin at 1 microM. These results are indicative of a common binding site for both CGRP and amylin in liver and skeletal muscle, and it is suggested that both peptides mediate their actions through the same effector system. The normal physiological importance and the relevance to the pathology of type 2 diabetes of these data are discussed.

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