Elsevier

Biochemical Pharmacology

Volume 66, Issue 11, 1 December 2003, Pages 2107-2115
Biochemical Pharmacology

Three receptor-activity-modifying proteins define calcitonin gene-related peptide or adrenomedullin selectivity of the mouse calcitonin-like receptor in COS-7 cells

https://doi.org/10.1016/j.bcp.2003.07.009Get rights and content

Abstract

Receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are heterodimeric complexes of the calcitonin-like receptor (CLR) together with associated receptor-activity-modifying proteins (RAMP)1, -2 or -3. The RAMP define the specificity of the CLR for CGRP or AM. Here, mouse (m)CLR/mRAMP1, -2 and -3 were expressed in COS-7 cells that lack detectable CGRP and AM receptors. myc epitope-tagged non-glycosylated mRAMP1 required V5-tagged mCLR for its translocation to the cell surface. The glycosylated myc-mRAMP2 and -3, on the other hand, were expressed at the cell surface in the absence of co-transfected mCLR. Selective binding of [125I]hαCGRP to mCLR/mRAMP1 expressing cells was inhibited by rat (r)αCGRP(1–37) and the CGRP antagonist rαCGRP(8–37) with ic50 of 7.0±1.6 nM and 1.0±0.1 nM (mean±SEM). rAM(1–50) and the AM antagonist rAM(20–50) inhibited [125I]hαCGRP binding at over 36-fold higher concentrations than rαCGRP. In mCLR/mRAMP2 expressing cells, selective [125I]rAM binding was inhibited by rAM(1–50) and -(20–50) with ic50 of 8.9±2.6 nM and 34±9 nM. rαCGRP(1–37) and -(8–37) displaced the binding at over 25-fold higher concentrations. mCLR/mRAMP3 expressing cells recognized both [125I]hαCGRP and -rAM. The ic50 of rAM and rαCGRP(8–37) ranged between 5.8 and 7.0 nM, and those of rαCGRP and rAM(20–50) were only 4- to 8-fold higher. rαCGRP and rAM stimulated and rαCGRP(8–37) and rAM(20–50) antagonized mCLR/mRAMP1, -2 and -3 mediated cAMP formation with relative potencies that reflected the observed CGRP and AM selectivity of the three receptor types. In conclusion, mCLR/mRAMP1 and -2 are CGRP- and AM-selective receptors, respectively, whereas mCLR/mRAMP3 is an AM/CGRP receptor.

Introduction

CGRP and AM are potent vasodilators and as a consequence hypotensive peptides [1], [2]. They exhibit limited structural similarities which include six amino acid ring structures, formed by a disulfide bridge between cysteine residues, followed by an alpha helix and amidated C-termini. As a result, the peptides crossreact to a different degree with the respective receptors in tissues and cell lines [3]. The corresponding CLR requires the interaction with one of three RAMP1, -2 and -3 for the recognition of CGRP and/or AM [4].

The amino acid sequences of the RAMP of man, mouse, rat, pig, and guinea pig have been revealed through molecular cloning [4], [5], [6]. The RAMP are single transmembrane domain proteins with an extracellular N-terminus of about 100 amino acids and an intracellular C-terminal tail of 10 amino acids. RAMP1, -2 and -3 exhibit approximately 30% amino acid sequence identity.

The CLR of man, rat, pig and cow have been characterized through co-expression with RAMP1, -2 or -3 [4], [5], [7], [8]. Co-expression of cloned CLR with RAMP in cell lines of different species revealed variable CGRP and AM selectivity of expressed receptors. The variability can be explained in part by the presence of endogenous CLR and/or RAMP in most of the cells examined (for review see [9]). Besides, a non-peptidic CGRP antagonist was active with human unlike rodent RAMP1/CLR [10]. This selectivity was determined by a tryptophan residue present in human but not in rodent RAMP1.

Human (h)αCGRP(8–37), unlike hAM(22–52) or rat (r)AM(20–50), is an antagonist at a CGRP receptor of human SK-N-MC neuroblastoma cells [11]. A particular AM receptor subtype expressed in rabbit aortic endothelial cells and in neuroblastoma × glioma hybrid NG108-15 cells is antagonized by hAM(22–52) but not by hαCGRP(8–37) [12], [13]. Another AM receptor subtype found in rat astrocytes is more potently antagonized by hαCGRP(8–37) than hAM(22–52) [13]. Several CGRP and AM receptors expressed from cDNA encoding CLR and the corresponding RAMP transfected into various cells remain to be examined as far as the interaction with the antagonists CGRP(8–37) and hAM(22–52) or rAM(20–50) are concerned.

In the present study, the mCLR co-expressed with mRAMP1, -2 or -3 was examined in COS-7 cells which do not express detectable endogenous CLR and RAMP. The mCLR/mRAMP1 was a CGRP receptor antagonized by rαCGRP(8–37). The mCLR/mRAMP2, on the other hand, was an AM receptor antagonized by rAM(20–50). A mixed-type AM/CGRP receptor more potently antagonized by rαCGRP(8–37) than by rAM(20–50) was revealed with the mCLR co-expressed with mRAMP3. Translocation to the cell surface of an mCLR with an N-terminal V5 epitope-tag did not require RAMP. myc-mRAMP2 and -3, unlike myc-mRAMP1, were recognized at the cell surface in the absence of co-expressed mCLR.

Section snippets

cDNA constructs for expression of epitope-tagged mRAMP and mCLR

The constructs encoding mRAMP1, -2 and -3 with an N-terminal myc-epitope (myc-mRAMP1, -2 and -3) were obtained as follows. DNA fragments encoding mRAMP1, -2 and -3 without the corresponding signal sequences were obtained by PCR from previously cloned cDNA [5]. The nucleotide sequences of the primer pairs were for mRAMP1 (5′-ATATCTCGAGTGCCGGGACCCTGACTATGGGACTC-3′ and 5′-ATATCTCGAGGAATTCCCAGGTTTAAGAATACTCTTTTAA-3′), for mRAMP2 (5′-AAAACTCGAGTCTCCGGAGTCCCTGAACCAATC-3′ and

Cell surface expression of V5-mCLR and myc-mRAMP1, -2 and -3

V5-mCLR and myc-mRAMP1, -2 and -3 immunofluorescent staining of non-permeabilized and saponin-permeabilized COS-7 cells indicated cell surface and total expression, respectively. V5-mCLR was expressed at the cell surface in the absence of co-transfected mRAMP1, -2 or -3 (Fig. 1, top panel). Cell surface expression of the V5-mCLR was approximately 50% of total expression in the absence and the presence of the RAMP (not shown). Moreover, the levels of the V5-mCLR at the cell surface in the

Discussion

Here we have functionally characterized homologous mCLR/mRAMP1, -2 and -3 CGRP and AM receptors in COS-7 cells. The mCLR has been characterized with mRAMP1 [18]. The human, rat, porcine and bovine CLR were mainly analyzed with RAMP from different species and in HEK cells with endogenous RAMP, CLR and calcitonin receptors that interfere with the analysis [9]. COS-7 cells do not express functional RAMP and CLR [15]. To this end, neither transfection of myc-mRAMP1, -2 or -3 or V5-mCLR alone

Acknowledgements

The technical assistance of B. Langsam is gratefully acknowledged. This study was supported by the Swiss National Science Foundation, the Kanton of Zurich and the Schweizerischer Verein Balgrist.

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