Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms

Abstract

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are related peptides with distinct pharmacological profiles. Calcitonin-receptor-like receptor (CRLR, now known as CL) can function as either an AM receptor or a CGRP receptor, when cotransfected with receptor-activity-modifying proteins (RAMPs) that define ligand-binding specificity. The aim of the present study was to determine the role of endogenously expressed CL (EndoCL) in generating endogenous AM and CGRP receptors. We raised anti-human CL antibody and identified microvascular endothelial cells (MVECs) as a major CL-expressing cell type in tissues by immunohistochemistry. Cultured MVECs continue to express EndoCL as well as fully active endogenous AM- and CGRP-sensitive receptors in vitro, as demonstrated by the ability of both peptides to induce migration and Akt phosphorylation. We therefore tested the hypothesis that endothelial EndoCL can interact with both AM and CGRP by examining receptor internalisation and desensitisation (loss of the ability to induce Akt phosphorylation). We found that agonist-mediated internalisation of EndoCL occurs in response to AM but not CGRP in MVECs. However, AM-induced EndoCL internalisation was blocked by antagonists of both AM and CGRP receptors: AM22-52 and CGRP8-37, respectively. Furthermore, AM-induced EndoCL internalisation resulted in desensitisation not only of AM but also of CGRP receptors. Finally, CGRP also induced desensitisation of both endogenous AM and CGRP receptors, but did not mediate EndoCL internalisation despite interaction with this receptor. Thus, EndoCL interacts with both AM and CGRP, and simultaneously acts as a receptor for both peptides (i.e acting as an endogenous AM/CGRP receptor) in endothelial cells. Interaction with either ligand is sufficient to induce EndoCL desensitisation to both AM and CGRP, but differential mechanisms are involved since only AM induces EndoCL internalisation. These novel findings regarding regulation of EndoCL function in endothelial cells are likely to be of importance in conditions where AM or CGRP levels are elevated, such as cardiovascular disease, diabetes and inflammation.

VEGF is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting

“…The HIF target adrenomedullin was shown to promote physiological and pathological angiogenesis in vitro and in animal models (49)(50)(51). ADM signals angiogenesis by binding calcitonin-receptor-like receptor, which is widely expressed on normal and hypoxic endothelial cells (49,52).…”

Expression of Terminally Glycosylated Calcitonin Receptor–Like Receptor in Uterine Leiomyoma: Endothelial Phenotype and Association with Microvascular Density

“…The human endothelial CL cDNA was cloned into the pcDNA3.1 expression vector and termed hCLpcDNA (23). Myc-RAMP2 cloned into pcDNA3.1 was a gift from D. Poyner (Aston University, Birmingham, United Kingdom) and was originally prepared by Dr. S. Foord and coworkers (GlaxoSmithKline, Stevenage, United Kingdom).…”

“…However, only recently, experimental evidence from in vitro models confirmed that endogenous GPCR CL expressed in human endothelial cells is essential in AM-induced angiogenesis (22,23). Endothelial CL is fully glycosylated (biologically active) and interacts with AM, playing a role as a functional AM-sensitive receptor in vitro (23). Anti-human CL (hCL) antibodies have been successfully used to block AMinduced endothelial migration and tube formation in vitro (22).…”

Effects of hyperbaric oxygen on vascular endothelial function in patients with slow coronary flow

“…NO and ET-1 are also considered biomarkers for vascular endothelial function. As the main diastolic vascular factor, CGRP interacts with its receptors on vascular endothelial cells, which in turn enhances intracellular calcium levels via the cyclic adenosine monophosphate pathway [9]. This leads to enhanced activity of NO synthase, which further increases the vasodilatory effects of NO.…”

“…To further investigate the potential mechanisms of HBO treatment on vascular endothelial functions, plasma levels of NO [8], ET-1 [8], CGRP [9] were analyzed, these were considered as biomarkers for vascular endothelial functions, and hsCRP, a maker of the inflammatory response [10,11]. There were no significant differences in plasma levels of NO, ET-1, CGRP and hsCRP between the groups before treatment.…”