The calcitonin receptor-like receptor/receptor activity-modifying protein 1 heterodimer can function as a calcitonin gene-related peptide-(8–37)-sensitive adrenomedullin receptor

doi:10.1016/S0014-2999(02)02184-2

European Journal of Pharmacology

Volume 450, Issue 3, 30 August 2002, Pages 237-243

https://doi.org/10.1016/S0014-2999(02)02184-2 Get rights and content

Abstract

The receptor activity-modifying protein (RAMP)/calcitonin receptor-like (CRL) receptor heterodimer is thought to function as a receptor for either a calcitonin gene-related peptide (CGRP) (CRL receptor/RAMP1) or adrenomedullin (CRL receptor/RAMP2 or -3), depending on the RAMP isoform present. We examined the receptor specificity of adrenomedullin-induced increases in cAMP in human embryonic kidney (HEK)293 cells coexpressing human CRL receptor and human RAMP1 or RAMP2. In cells expressing CRL receptor/RAMP1, adrenomedulin-induced increases in cAMP were comparable to those induced by α-CGRP, and the CGRP receptor antagonist α-CGRP-(8–37), but not the adrenomedullin receptor antagonist adrenomedullin-(22–52), blocked the adrenomedullin-evoked responses. Cells expressing CRL receptor/RAMP2 responded more selectively to adrenomedullin; in this case, the effect was blocked by adrenomedullin-(22–52) but not by α-CGRP-(8–37). Real-time quantitative polymerase chain reaction confirmed that cotransfection of CRL receptor and RAMP1 had no effect on the endogenous expression of RAMP2. Thus, CRL receptor/RAMP1 likely functions as an adrenomedullin receptor as well as a CGRP receptor, which may explain why many of the actions of adrenomedullin are potently antagonized by α-CGRP-(8–37).

Introduction

Calcitonin gene-related peptide (CGRP) and adrenomedullin belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators Brain et al., 1985, Kitamura et al., 1993. CGRP is a 37-amino acid neuropeptide present throughout the central and peripheral nervous systems (Wimalawansa, 1997). Adrenomedullin, by contrast, is comprised of 52 amino acids and acts as an autocrine/paracrine agent in a variety of tissues, though best understood is its role as a local regulator of cardiovascular function (Eto, 2001). Both peptides induce production of cAMP and Ca²⁺ mobilization in a variety of cell types Shimekake et al., 1995, Drissi et al., 1998.

Since its discovery, adrenomedullin has been shown to share a number of pharmacological features with CGRP. For instance, many effects of adrenomedullin are blocked by α-CGRP-(8–37), a selective CGRP receptor antagonist (Chiba et al., 1989) state regarding the pharmacological features of α-CGRP-(8–37). Meanwhile Nuki et al. and the listed authors demonstrate the effects of adrenomedullin that are blocked by the α-CGRP-(8–37) Nuki et al., 1993, Nishimura and Suzuki, 1997, Okamura et al., 1997, Yoshimoto et al., 1998. Moreover, α-CGRP-(8–37), but not adrenomedullin-(22–52), a selective adrenomedullin receptor antagonist (Eguchi et al., 1994), blocks adrenomedullin-induced cellular responses Nishikimi et al., 1998, Hasbak et al., 2001, Tomoda et al., 2001. These findings suggest that adrenomedullin binds not only to its specific receptors, but also to receptors that bind both CGRP and adrenomedullin.

Receptor activity-modifying proteins (RAMPs) are a recently identified group of accessory proteins that serve to transport calcitonin receptor-like (CRL) receptors to the cell surface, where they form functional CGRP and adrenomedullin receptors (McLatchie et al., 1998). The three RAMP isoforms (RAMP1, RAMP2 and RAMP3) are each comprised of approximately 160 amino acids, making up a large extracellular N-terminal domain, a single membrane-spanning domain and a short cytoplasmic domain. Coexpression of RAMP2 or RAMP3 with CRL receptor leads to both proteins being presented at the plasma membrane as a heterodimeric adrenomedullin receptor, while coexpression of CRL receptor with RAMP1 enables the resultant heterodimer to function as a CGRP receptor McLatchie et al., 1998, Muff et al., 1998, Fraser et al., 1999, Kuwasako et al., 2000. Although α-CGRP has far less affinity than adrenomedullin for CRL receptor/RAMP2 and CRL receptor/RAMP3, the affinity of adrenomedullin for CRL receptor/RAMP1 is comparable to that of α-CGRP Fraser et al., 1999, Kamitani et al., 1999, Kuwasako et al., 2000, Kuwasako et al., 2001, Oliver et al., 2001. In addition, in cells expressing much higher levels of CRL receptor and RAMP1 Hasbak et al., 2001, Tomoda et al., 2001 than RAMP2 and -3, the effects of adrenomedullin are blocked by α-CGRP-(8–37), but not by adrenomedullin-(22–52). Taken together, these results suggest that adrenomedullin-induced cellular responses are mediated via the CRL receptor/RAMP1 complex. To confirm this, we cotransfected human CRL receptor and human RAMP1 or RAMP2 into HEK293 cells, which otherwise express no functional CGRP or adrenomedullin receptors, and examined the effects of α-CGRP-(8–37) and adrenomedullin-(22–52) on adrenomedullin-evoked cAMP production in the transfected cells.

Section snippets

Materials

A plasmid containing human CRL receptor cDNA (Fluhmann et al., 1995) was a kind gift from Dr. Kenji Kangawa (National Cardiovascular Research Institute, Osaka, Japan). Human α-CGRP, adrenomedullin, α-CGRP-(8–37) and adrenomedullin-(22–52) were purchased from the Peptide Institute (Osaka, Japan). All other chemicals were of reagent grade or higher and were obtained from various commercial suppliers.

Expression constructs

Human CRL receptor, RAMP1 and RAMP2 were modified to provide a consensus Kozak sequence as

Ligand-induced cAMP production in HEK293 cells coexpressing human CRL receptor and human RAMP1 or -2

The EC₅₀ for α-CGRP-and adrenomedullin-induced cAMP production in intact HEK293 cells was >2.0×10⁻⁶ M for both agonists, which is indicative of the absence of functional CGRP and adrenomedullin receptors in this cell type (Fig. 1A). In HEK293 cells transfected with RAMP1, however, CGRP elicited eightfold increases in cAMP over the levels seen in untransfected cells (data not shown), suggesting that RAMP1 and endogenous calcitonin receptors are capable of forming a functional CGRP receptor

Discussion

We demonstrated that in HEK293 cells coexpressing human CRL receptor and human RAMP1, adrenomedullin elicited concentration-dependent increases in the levels of intracellular cAMP that were comparable to those elicited by α-CGRP and that were blocked by α-CGRP-(8–37) but not by adrenomedullin-(22–52). Consistent with these findings, there was no remarkable difference in the EC₅₀ values for adrenomedullin-evoked cAMP production mediated via CRL receptor/RAMP1 and CRL receptor/RAMP2. By contrast,

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The calcitonin receptor-like receptor/receptor activity-modifying protein 1 heterodimer can function as a calcitonin gene-related peptide-(8–37)-sensitive adrenomedullin receptor

Abstract

Introduction

Section snippets

Materials

Expression constructs

Ligand-induced cAMP production in HEK293 cells coexpressing human CRL receptor and human RAMP1 or -2

Discussion

J. Biol. Chem.

Eur. J. Pharmacol.

J. Biol. Chem.

Peptides

Biochem. Biophys. Res. Commun.

Eur. J. Pharmacol.

Mol. Cell. Endocrinol.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Mol. Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Eur. J. Pharmacol.

Biochem. Biophys. Res. Commun.

Eur. J. Pharmacol.

J. Biol. Chem.

Neurosci. Lett.