Functional Expression of Heteromeric Calcitonin Gene-related Peptide and Adrenomedullin Receptors in Yeast

Abstract

The ability of G protein-coupled receptors (GPCRs) to form homo-and heteromeric complexes has important implications for the regulation of cellular events. A no table example G protein-coupled receptors (GPCRs)1 represent the largest family of cell-surface receptors. As key controllers of diverse physiological processes, they are of considerable biological and therapeutic interest. Although this class of receptors was originally thought to act as monomers that mediate their effects through stimulation of heterotrimeric G proteins, recent studies indicate that they can also act as multimeric complexes that, besides G proteins, can directly regulate a variety of other downstream effectors, including mitogen-activated protein kinase cascades and transcription factors (1). Several early lines of evidence have suggested that certain families of GPCRs can form homo-oligomers (2, 3). More recent evidence (3-8) has confirmed this and has indicated that GPCRs can also form heteromeric complexes, either with related GPCRs or with members of distinct families of GPCRs. Moreover, some receptors appear to require interaction with additional accessory factors for proper function (9 -12). GPCRs therefore seem to function in a rather complex molecular environment. One striking example of the complexity of some GPCRs is provided by receptors for adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP), two members of the calcitonin family of peptides. In this case, a seven-transmembrane protein, the calcitonin receptor-like receptor (CRLR), has been reported to require two different types of associated proteins, receptor activity modifying proteins (RAMPs) and receptor component protein (RCP), for full activity (13,14).CGRP is a potent vasoactive neuropeptide, which has been implicated in vasodilation, migraine, and chronic pain, whereas ADM is a multifunctional regulatory peptide with a wide range of biological actions, including vasodilation, cell growth, natriuresis, and certain antimicrobial effects (15,16). Despite the physiological importance and clinical implications of these two peptides, the identification and characterization of their receptors have been difficult for some time. A seventransmembrane protein with 55% homology to the calcitonin receptor (CRLR) provided CGRP receptor function only after transfection into specific cellular backgrounds (17, 18). The lack of a CGRP response in other cell lines made positive identification of this protein as a CGRP receptor problematic and suggested that CRLR might require cell type-specific accessory factors to become CGRP-responsive. A novel accessory protein, RAMP1, was identified that generated a functional CGRP receptor upon co-transfection with CRLR into cell culture (19). Confirming the idea that cell type-specific co-factors are required for CGRP responsiveness, transfection of CRLR into cell lines only yielded a functional CGRP receptor when the cell line endogenously expresses RAMP1. Conversely, transfection with RAMP1 only resulted in CGRP receptor function...

Decrease in arterial pressure induced by adrenomedullin in the hypothalamic paraventricular nucleus is mediated by nitric oxide and GABA

“…This 52-amino acid peptide is a member of the calcitonin peptide family, which also includes calcitonin, calcitonin gene-related peptide (CGRP), and amylin. Both ADM and its receptor system, which is composed of a calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) 2 or 3 [2][3][4][5][6][7], have been demonstrated in peripheral tissues and in the central nervous system (CNS) [8,9]. In the CNS, high levels of ADM and its receptors have been found in the hypothalamus [10], especially in the paraventricular nucleus (PVN) [11,12].…”

Expression of human melanocortin 4 receptor in Saccharomyces cerevisiae

“…However, so far the GPCRs that bind small molecules as agonists have usually been explored [27,30,31]. For the analysis of GPCRs, which bind larger peptides, co-expression of ligand and receptor in the same yeast cells has been used [26,29]. The extracellular activation of the V2 vasopressin receptor with the 9-amino acid vasopressin peptide has been reported for the peptide binding GPCRs in yeast cells [28].…”

“…Expression of GPCRs in yeast Saccharomyces cerevisiae, which is engineered to couple with the mammalian GPCRs and requires activation of the receptor for growth, has been used to pharmacologically characterize a number of various receptors [26][27][28][29][30][31]. The advantages of such an approach in GPCR research include low cost propagation, automation of the screening and a low level of false positive results that are common in mammalian cells due to endogenous expression of ligands or related receptors.…”

Adrenomedullin Selectivity of Calcitonin-like Receptor/Receptor Activity Modifying Proteins

“…A similar receptor may exist in Swiss 3T3 mouse fibroblasts, in which CGRP(8-37) and hAM were equipotent antagonists with a Ki of 130 nmol/l (28). In certain tissues, the AM response is antagonized by CGRP(8-37) and not by hAM(22-52) (21,(28)(29)(30)(31). In these tissues, AM presumably interacts with the CGRP1 receptor defined as the CL receptor associated with RAMP1.…”

“…These results are in accordance with the 10-100-fold higher potency of hAM(22-52) over CGRP in AM-binding inhibition. The AM response was also antagonized by hAM(22-52), but not by CGRP , in yeast cells transfected with a pCL receptor and pRAMP2 (21). For the rCL receptor/RAMP2, no Ki values are available, although antagonism of rAM (20-50) is predicted based on the observed IC50.…”