Associate editor: J. Wess
GPCR modulation by RAMPs

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Abstract

Our conceptual understanding of the molecular architecture of G-protein coupled receptors (GPCRs) has transformed over the last decade. Once considered as largely independent functional units (aside from their interaction with the G-protein itself), it is now clear that a single GPCR is but part of a multifaceted signaling complex, each component providing an additional layer of sophistication. Receptor activity-modifying proteins (RAMPs) provide a notable example of proteins that interact with GPCRs to modify their function. They act as pharmacological switches, modifying GPCR pharmacology for a particular subset of receptors. However, there is accumulating evidence that these ubiquitous proteins have a broader role, regulating signaling and receptor trafficking. This article aims to provide the reader with a comprehensive appraisal of RAMP literature and perhaps some insight into the impact that their discovery has had on those who study GPCRs.

Introduction

Receptor activity-modifying proteins (RAMPs) form a small family of 3 single-transmembrane spanning proteins that can form complexes with G-protein coupled receptors (GPCRs), altering their trafficking, pharmacology, and/or signaling capabilities. The interactions of RAMPs with the calcitonin (CT) receptor-like receptor (CL) are the most extensively studied. These experiments have identified that CL/RAMP complexes are stable dimers that form and are processed in the endoplasmic reticulum/Golgi and stay in this form during transport to the cell surface, peptide interaction and receptor activation, internalization, and subsequent recycling or degradation. However, the RAMP subtype appears to affect the trafficking properties of the receptor complex, being governed by specific protein–protein interactions with the RAMP intracellular C-terminus.

While RAMPs provide a mechanism for inducing diversity in receptor repertoire for the calcitonin family of peptides, there is now enticing evidence to suggest that these proteins have a much broader role. Indeed, they are ubiquitous proteins and may be found where calcitonin peptide family receptors are not. Investigation of the functional consequences of the novel interaction of RAMP2 with the vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide 1 (VPAC1) receptor has indicated a novel role for RAMPs in the modulation of receptor signaling. RAMPs also interact with other family B GPCRs, but the significance of these interactions has yet to be determined.

Section snippets

Discovery

RAMPs were first identified during attempts to expression clone a receptor for the neuropeptide calcitonin gene-related peptide (CGRP; McLatchie et al., 1998). Historical evidence had suggested that CGRP acted through a GPCR, as its binding had proven sensitive to GTP analogues and stimulation of various tissues and cells led to the accumulation of cAMP, suggesting activation of a Gs-coupled GPCR. However, attempts to clone such a receptor proved difficult. A putative canine CGRP receptor,

Primary sequence

All RAMPs are built around a common structure and form a small family of proteins, each possessing a single transmembrane α-helix, an extracellular amino terminus, and a short intracellular C-terminus. Three human RAMPs have been cloned, RAMP1, RAMP2 (both cloned from SK-N-MC cells), and RAMP3 (cloned from human spleen; McLatchie et al., 1998). The sequences of the known RAMPs are illustrated in Fig. 1. Full RAMP sequences are now available from 9 species, with partial sequence data from

Calcitonin gene-related peptide/AM receptor paradigm

The classic function attributed to RAMPs is their ability to switch the pharmacology of CL, thus providing a novel mechanism for modulating receptor specificity. Thus, the CL/RAMP1 complex is a high affinity CGRP receptor, but in the presence of RAMP2, CL specificity is radically altered, the related peptide AM being recognized with the highest affinity and the affinity for CGRP being reduced ∼ 100-fold. CL/RAMP3 receptors are interesting in that while AM is the highest affinity peptide, CGRP is

General summary of distribution

Overall, RAMPs have a relatively ubiquitous distribution with at least 1 RAMP identified (at least by PCR) in each tissue or cell line that has been studied (Sexton et al., 2001). RAMP1 is expressed in many tissues, including the heart, uterus, brain, bladder, pancreas, skeletal muscle, and gastrointestinal tract. RAMP2 is highly expressed in the lung and also in the heart, placenta, skeletal muscle, and pancreas. RAMP3 has a widespread distribution in the human but appears to have a more

Receptor activity-modifying proteins and signaling

Some consideration has been given to the potential role that RAMPs may have in modifying receptor behaviors other than ligand binding pharmacology. An additional functional consequence might be that of alteration of receptor signaling characteristics.

Receptor activity-modifying proteins as chaperones

For CL, heterodimerization with a RAMP is a prerequisite for its efficient transport to the cell surface. In this sense, RAMPs have a chaperone role (McLatchie et al., 1998). Epitope tagging of CL has yielded valuable information regarding its transport to the cell surface and shows that each RAMP is able to elicit translocation of CL from intracellular compartments to the cell surface. Fluorescence-associated cell sorting data and images from confocal microscopy show that there is a much

G-protein coupled receptors

At present, there is only limited information regarding the interaction of RAMPs with receptors outside of the calcitonin receptor family. The most convincing argument for a broader role for RAMPs than just modulating receptor specificity for the calcitonin peptide family was a recent publication investigating potential interactions of RAMPs with other family B GPCRs (Fig. 4). An inherent property of RAMPs is their inefficient transport to the cell surface in the absence of an interacting

Receptor activity-modifying protein specificity

The analysis of RAMP interaction with family B receptors provides clear evidence for specificity in the interaction profile of individual RAMPs. CL, calcitonin, and VPAC1 receptors can interact with all 3 RAMPs, whereas glucagon and PTH1 receptors appear to interact only with RAMP2, and PTH2 receptors, only with RAMP3 (Christopoulos et al., 2003). For the family C calcium sensing receptor, there appears to be functional interaction with RAMP1 or RAMP3 but not RAMP2.

For those receptors capable

Receptor activity-modifying protein regulation (in disease/pathophysiological states)

There has been much interest in the dynamic regulation of RAMPs. This has most commonly been investigated in animal models of disease where RAMP mRNA expression may be differentially regulated. Many of these studies have recently been summarized in comprehensive tabular format in 2 reviews (Kuwasako et al., 2004, Udawela et al., 2004). Most of the work so far has focused on the AM receptor system (CL/RAMP2), with particular emphasis on the potentially important cardiovascular role for AM, and

Concluding remarks

RAMPs transformed our understanding of how receptor pharmacology can be modulated and provided a novel mechanism for generating receptor subtypes within a subset of family B GPCRs. Their role has now broadened and they have been shown to interact with several other family B GPCRs, in 1 case modifying signaling parameters. There is now evidence to suggest that their interactions also reach into family C, and possibly family A, GPCRs, indicating that their function may not be restricted to

Acknowledgments

PMS is a Senior Research Fellow of the National Health and Medical Research Council of Australia. DLH is supported by the Lottery Health Commission (New Zealand), Auckland Medical Research Foundation, and the University of Auckland Staff Research Fund. DRP was supported by the BBSRC. Elements of some figures were created using templates from Science Slides (VisiScience, NC, USA).

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