Pharmacological Discrimination of Calcitonin Receptor: Receptor Activity-Modifying Protein Complexes

Abstract

Calcitonin (CT) receptors dimerize with receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) receptors, but there is no reliable means of pharmacologically distinguishing these receptors. We used agonists and antagonists to define their pharmacology, expressing the CT (a) receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of ␣CGRP, being a weak agonist at CT (a) , AMY 2(a) , and AMY 3(a) receptors but considerably more potent at AMY 1(a) receptors. Likewise, the linear calcitonin gene-related peptide (CGRP) analogs (Cys(ACM) 2,7 )h␣CGRP and (Cys(Et) 2,7 )h␣CGRP were only effective at AMY 1(a) receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY 2(a) receptor phenotype; thus, AMY 2(a) was not examined further in this study. The antagonist peptide salmon calcitonin 8-32 (sCT 8-32 ) did not discriminate strongly between CT and AMY receptors; however, AC187 was a more effective antagonist of AMY responses at AMY receptors, and AC413 additionally showed modest selectivity for AMY 1(a) over AMY 3(a) receptors. CGRP 8-37 also demonstrated receptor-dependent effects. CGRP 8-37 more effectively antagonized AMY at AMY 1(a) than AMY 3(a) receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT (a) receptor. Low CGRP 8-37 affinity and agonism by linear CGRP analogs at AMY 1(a) are the classic signature of a CGRP 2 receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT (a) , AMY 1(a) , and AMY 3(a) receptors, providing a means to delineate the physiological significance of these receptors.

Amyloid β (Aβ) Peptide Directly Activates Amylin-3 Receptor Subtype by Triggering Multiple Intracellular Signaling Pathways

“…However, due to the lack of selective pharmacological tools, significant complexity of this system and lack of specific RAMP antibodies, it has been difficult to confidently assign specific amylin functions to one of these receptor subtypes. Data from functional bioassays, including binding studies (16,29,30), demonstrate that AMY3, which is a heterodimeric complex of CTR and RAMP3, has a high affinity for amylin. Amylin receptor components (CTR and RAMPs) are reported to be distributed widely in the central nervous system with pronounced expression within spinal cord, brain stem, cortex, hypothalamus, and hippocampus (31,32).…”

“…The CTR component of this receptor shares the same general GPCR architecture: seven membrane-spanning interconnected ␣-helices that transmit extracellular signals to the intracellular cytoplasmic signaling cascade. Activation of amylin receptors in mammalian cells has been shown previously to raise cAMP and presumed to involve G S protein (Gs ␣-guanine nucleotide-binding signal transduction protein) and stimulation of adenylate cyclase (16,17).…”

Targeting CGRP: A New Era for Migraine Treatment

“…The complex CTR/RAMP1 is classified as an amylin receptor, which explains the name AMY1, but has also a high affinity for CGRP and consequently is considered a dual CGRP/amylin receptor [68,69]. Both receptors, CGRP and AMY1, are strongly and equally represented in rat TG neurons since equipotent results are found when antagonizing each of them separately.…”

Gut hormones such as amylin and GLP-1 in the control of eating and energy expenditure

“…[52][53][54] Receptor binding and mapping studies have shown a wide distribution of the amylin receptor components throughout the central nervous system, and a high density of both the CTR and RAMPs is found in the AP. [55][56][57][58] Recent experiments in our laboratory have shown that single amylinactivated AP neurons contain all necessary components of the functional amylin receptor 1 or 3, that is, CTR plus RAMP1 or CTR plus RAMP3, respectively; in fact, AP neurons may often contain both types of RAMPs within single cells.…”