From Micromolar to Nanomolar Affinity:  A Systematic Approach To Identify the Binding Site of CGRP at the Human Calcitonin Gene-Related Peptide 1 Receptor†

1998 ◽  
Vol 41 (1) ◽  
pp. 117-123 ◽  
Author(s):  
Beate Rist ◽  
Michael Entzeroth ◽  
Annette G. Beck-Sickinger
Keyword(s):  
Binding Site ◽  
Systematic Approach ◽  
Calcitonin Gene Related Peptide ◽  
Human Calcitonin ◽  
Related Peptide ◽  
Calcitonin Gene

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

1991 ◽  
Vol 277 (1) ◽  
pp. 139-143 ◽  
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.

scholarly journals Calcitonin gene-related peptide receptor antagonist human CGRP-(8-37)

1989 ◽  
Vol 256 (2) ◽  
pp. E331-E335 ◽  
Author(s):  
T. Chiba ◽  
A. Yamaguchi ◽  
T. Yamatani ◽  
A. Nakamura ◽  
T. Morishita ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Calcitonin Gene Related Peptide ◽  
Human Calcitonin ◽  
Liver Plasma Membrane ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Liver Membranes ◽  
Calcitonin Receptors ◽  
Stimulation Of

From this study, we predicted that the human calcitonin gene-related peptide (hCGRP) fragment hCGRP-(8-37) would be a selective antagonist for CGRP receptors but an agonist for calcitonin (CT) receptors. In rat liver plasma membrane, where CGRP receptors predominate and CT appears to act through these receptors, hCGRP-(8-37) dose dependently displaced 125I-[Tyr0]rat CGRP binding. However, hCGRP-(8-37) had no effect on adenylate cyclase activity in liver plasma membrane. Furthermore, hCGRP-(8-37) inhibited adenylate cyclase activation induced not only by hCGRP but also by hCT. On the other hand, in LLC-PK1 cells, where calcitonin receptors are abundant and CGRP appears to act via these receptors, the bindings of 125I-[Tyr0]rat CGRP and 125I-hCT were both inhibited by hCGRP-(8-37). In contrast to liver membranes, interaction of hCGRP-(8-37) with these receptors led to stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production in LLC-PK1 cells, and moreover, this fragment did not inhibit the increased production of cAMP induced not only by hCT but also by hCGRP. Thus hCGRP-(8-37) appears to be a useful tool for determining whether the action of CGRP as well as that of CT is mediated via specific CGRP receptors or CT receptors.

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

1988 ◽  
Vol 50 (2) ◽  
pp. 480-485 ◽  
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

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

1996 ◽  
Vol 318 (1) ◽  
pp. 241-245 ◽  
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.

Synthetic Human Calcitonin Gene Related Peptide in Action

2010 ◽  
Vol 16 (8) ◽  
pp. S73
Author(s):  
Asad Sheikh ◽  
James Vacek ◽  
Jeff Southard ◽  
Pamela Gayheart-Walsten ◽  
Ty Speece ◽  
...  
Keyword(s):  
Calcitonin Gene Related Peptide ◽  
Human Calcitonin ◽  
Related Peptide ◽  
Calcitonin Gene

Effects of galanin and calcitonin gene-related peptide on insulin and glucagon secretion in man

1990 ◽  
Vol 123 (6) ◽  
pp. 591-597 ◽  
Author(s):  
Bo Ahrén
Keyword(s):  
Insulin Secretion ◽  
Plasma Insulin ◽  
Glucagon Secretion ◽  
Calcitonin Gene Related Peptide ◽  
Human Calcitonin ◽  
C Peptide ◽  
Related Peptide ◽  
Insulin And Glucagon Secretion ◽  
Calcitonin Gene ◽  
Human Volunteers

Abstract. To study the influence of the pancreatic neuropeptides, galanin and calcitonin gene-related peptide, on insulin and glucagon secretion in man, synthetic porcine galanin (80 pmol·kg−1·min−1; N=6) or synthetic human calcitonin gen-related peptide (10 pmol·kg−1·min−1; N = 5) was infused intravenously in human volunteers. Following 5 min of infusion, arginine (5 g bolus + 10 mg·kg−1·min−1) was given. Galanin did not affect basal or arginine-stimulated insulin secretion judged from determinations of plasma insulin and C-peptide. Similarly, galanin did not affect arginine-stimulated glucagon secretion. Calcitonin gene-related peptide did not affect basal or arginine-stimulated insulin or glucagon secretion. However, calcitonin gene-related peptide slightly potentiated the arginine-induced hyperglycemia (p<0.01). Thus, in man, galanin has no influence on insulin or glucagon secretion when infused at 80 pmol·kg−1·min−1, whereas CGRP at 10 pmol·kg−1·min−1 induces slight hyperglycemia.

Human calcitonin gene related peptide: a potent endogenous vasodilator in man

1986 ◽  
Vol 70 (4) ◽  
pp. 389-393 ◽  
Author(s):  
A. D. Struthers ◽  
M. J. Brown ◽  
D. W. R. Macdonald ◽  
J. L. Beacham ◽  
J. C. Stevenson ◽  
...  
Keyword(s):  
Diastolic Pressure ◽  
Normal Subjects ◽  
Calcitonin Gene Related Peptide ◽  
Plasma Calcium ◽  
Human Calcitonin ◽  
High Concentration ◽  
Plasma Adrenaline ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Perivascular Nerves

1. In addition to calcitonin and katacalcin, it is now known that the human calcitonin gene encodes a novel peptide called calcitonin gene related peptide (CGRP). In experimental animals, CGRP produces vasodilatation and complex changes in plasma calcium. 2. We have now assessed its biological activity in man by infusing human CGRP (hCGRP) into six normal volunteers. hCGRP (545 pmol/min) caused the diastolic pressure to fall from 64 ± 5 to 55 ± 7mmHg (P < 0.05), the heart rate to increase from 61 ± 7 to 87 ± 5 beats/min (P < 0.05) and the skin temperature to increase from 33.7 ± 0.9 to 34.9 ± 0.5°C. Plasma noradrenaline increased from 481 ± 126 to 835 ± 65 pg/ml (P < 0.05) and plasma adrenaline from 57 ± 17 to 82 ± 12 pg/ml (P < 0.05). There were no significant changes in the albumin-corrected plasma calcium. 3. hCGRP is thus a potent endogenous vasodilator in man and is in fact more potent than any other known vasodilator. Together with the observations that CGRP circulates in normal subjects at relatively high concentration (approximately 25 pmol/l) and that CGRP is present in perivascular nerves, this study suggests a possible role for CGRP in controlling peripheral vascular tone in man.

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