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 notable example of heteromer formation is the interaction of the calcitonin receptor-like receptor (CRLR) with different members of the receptor activity modifying protein (RAMP) family, which results in the formation of two different receptors, a calcitonin gene-related peptide (CGRP) receptor and an adrenomedullin receptor. To analyze the role of RAMPs in determining ligand specificity, we have co-expressed CRLR and RAMP proteins in the yeast Saccharomyces cerevisiae, which provides a null system to study the function of mammalian receptors. Co-expression of RAMP1 and CRLR reconstituted a CGRP receptor that was able to activate the pheromone-signaling pathway with pharmacological properties similar to those observed previously in mammalian cells. Co-expression of CRLR with RAMP2 or RAMP3 resulted in a response with the pharmacological properties of an adrenomedullin receptor. These data indicate that RAMPs are necessary and sufficient to determine ligand specificity of CRLR. Contrary to observations in mammalian cells, the glycosylation of CRLR was not affected by the presence of RAMPs in yeast, indicating that glycosylation of CRLR is not the prime determinant of ligand specificity. The first functional reconstitution of a heteromeric seven transmembrane receptor in yeast suggests this organism as a useful research tool to study the molecular nature of other heteromeric receptors.
Highlights
G protein-coupled receptors (GPCRs)1 represent the largest family of cell-surface receptors
A notable example of heteromer formation is the interaction of the calcitonin receptor-like receptor (CRLR) with different members of the receptor activity modifying protein (RAMP) family, which results in the formation of two different receptors, a calcitonin gene-related peptide (CGRP) receptor and an adrenomedullin receptor
Co-expression of CRLR and RAMP1 in Yeast Is Sufficient for a CGRP Response—Haploid S. cerevisiae cells contain a cell type-specific seven-transmembrane receptor (Ste2 in MATa cells or Ste3 in MAT␣ cells) coupled to a heterotrimeric G protein that consists of a G␣ subunit (GPA1), a G subunit (STE4), and a G␥ subunit (STE18)
Summary
G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. More recent evidence [3,4,5,6,7,8] has confirmed this and has indicated that GPCRs can form heteromeric complexes, either with related GPCRs or with members of distinct families of GPCRs. some receptors appear to require interaction with additional accessory factors for proper function (9 –12). 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]. A heterologous expression system that lacks endogenous expression of both RCPs and RAMPs might be useful to study the contribution of the various accessory proteins to the pharmacology of the CGRP and ADM receptors
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