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Modulating receptor function through RAMPs: can they represent drug targets in themselves?

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G protein-coupled receptors (GPCRs) are successfully exploited as drug targets. As our understanding of how distinct GPCR subtypes can be generated expands, so do possibilities for therapeutic intervention via these receptors. Receptor activity-modifying proteins (RAMPs) are excellent examples of proteins that enhance diversity in GPCR function. They facilitate the creation of binding pockets, controlling the pharmacology of some GPCRs. Moreover, they have the ability to regulate cell-surface trafficking, internalisation and signalling of GPCRs, creating novel opportunities for drug discovery. RAMPs could be directly targeted by drugs, or advantage could be taken of unique RAMP/GPCR interfaces for generating highly selective ligands.

Introduction

It is generally well accepted that the largest group of ‘druggable’ proteins is represented by a handful of cell-surface receptor superfamilies. Of these, G protein-coupled receptors (GPCRs) constitute the largest single grouping, yet the number of actively targeted GPCRs still represents only a small fraction of the total number of GPCRs that could be effectively exploited therapeutically. These receptors, which signal predominantly through heterotrimeric G proteins, comprise ∼2% of the human genome and represent the major signalling system in cells. Mammalian GPCRs are further subdivided into three groups: Family A, the largest family, which contains receptors for prototypical neurotransmitters and hormones such as the biogenic amines; Family B, incorporating peptide hormone receptors such as calcitonin (CT), glucagon and secretin receptors; and Family C, including receptors for small molecules such as glutamate, GABA and calcium. GPCR-based signalling complexes engender enormous diversity and amplification in cell signalling processes and this is likely to account for the ubiquitous distribution of GPCRs across all cell types. Moreover, it is not surprising that many major disease states, such as CNS disorders, metabolic disorders and cardiovascular disease, often involve aberrations in GPCRs or their signalling pathways [1].

Increasing evidence indicates that GPCRs, like many other signalling proteins, can form oligomeric protein arrays and that these are crucial to many aspects of GPCR function, including cellular trafficking, compartmentalisation and signalling, regulation and even ligand recognition. It is thought that, for many GPCRs, constitutive dimers or oligomers act as the core functional unit, but the GPCR, either as a monomer, or oligomeric complex, may also interact with a diverse range of other proteins that regulate their function. These interactions create novel opportunities for drug discovery and development. An exemplar model for protein–protein modulation of GPCR function is the receptor activity-modifying protein (RAMP) family.

RAMPs are a family of three type I transmembrane proteins initially shown to regulate the glycosylation, transport and pharmacological phenotype of the, then orphan, CT receptor-like (CL, Box 1) receptor (CLR) [2]. In the absence of RAMPs, the CLR is principally retained intracellularly, however, RAMP interaction with the receptor leads to terminal glycosylation of the receptor and transit of the complex from the ER/Golgi to the cell surface. Similarly RAMPs, in the absence of an interacting receptor partner, are relatively poorly expressed at the cell surface and are translocated in complex with the receptor to the cell surface 2, 3. In addition to this chaperone role, RAMPs in stable complex with the CLR are required for the expression of phenotype, whereby RAMP1/CLR is a CT gene-related peptide (CGRP) receptor, while RAMP2/CLR and RAMP3/CLR exhibit distinct adrenomedullin (AM) receptor phenotypes (see below) 4, 5, 6, 7. Thus, the discovery of RAMPs revealed a novel mechanism for producing phenotypic diversity in receptor response. Subsequently, RAMPs were shown to interact with the related CT receptor, and each RAMP/CT receptor complex displayed a distinct amylin receptor phenotype 8, 9.

For the CT receptor and CLR, strong interaction occurs for all three RAMPs. These receptors are family B peptide hormone receptors, and subsequent analysis of other members of this receptor family for RAMP interaction, utilizing an assay of translocation of RAMPs to the cell surface, revealed additional RAMP-receptor partners with different degrees of specificity in the RAMP-receptor interaction. Like the CT receptor and CLR, the VPAC1 receptor interacted with all three RAMPs, whereas the PTH1 and glucagon receptors interacted specifically with RAMP2, and the PTH2 receptor specifically with RAMP3 [3]. No measurable translocation of RAMPs was seen, however, with the VPAC2, GHRH, GLP-1 or GLP-2 receptors, although this does not exclude the potential for weak or non-trafficking interactions between the receptors and RAMPs. With the exception of the VPAC1/RAMP2 interaction (see below), little is known of the potential role of RAMPs in the function for these receptors. More recently, a chaperone role for RAMPs 1 and 3, but not RAMP2, in the trafficking of the Family C, calcium sensing receptor (CaS receptor) has been reported [10], illustrating potential for RAMP modulation of receptors outside of Family B GPCRs. Although individual RAMPs are differentially expressed, the protein family is very broadly distributed throughout cells and organs of the body, beyond the localisation of known receptor partners (reviewed in 6, 11, 12), providing significant scope for additional RAMP-interacting GPCRs to be discovered. The full extent of RAMP interaction with other GPCRs, however, is yet to be elucidated.

Thus, RAMPs have the potential to interact with, and modulate the function of many GPCRs (Figure 1). This offers access to novel strategies for drug development at these highly tractable drug targets.

Section snippets

RAMP-regulated GPCR pharmacology

The most well-studied consequence of RAMP interaction with GPCRs is their ability to alter pharmacology; they function as pharmacological switches for the CT peptide family [7]. This family comprises CT, amylin, CGRP, AM and AM2 (intermedin). These peptides have many biological activities and some of their receptors are validated pharmaceutical targets for diseases including diabetes, migraine (see below) and osteoporosis. As indicated above, the receptors for these peptides are two Family B

RAMP-regulated receptor signalling

There is increasing evidence that RAMPs can play a role in the signalling profile of receptors. This has been most extensively studied for RAMP/CT receptor-derived AMY receptors, where early data identified receptor isoform-dependent and cell background-dependent differences in the ability of RAMP2 to create a high affinity AMY phenotype. In COS-7, and rabbit aortic endothelial cells, RAMP2 only weakly engenders amylin binding from the CT(a) receptor isoform, but strongly induces an AMY

RAMP-regulated receptor trafficking

One of the original proposed functions for RAMPs was to act as chaperones, to promote cell-surface expression of the CLR [2]. All three RAMPs can associate with this receptor in the endoplasmic reticulum and promote terminal glycosylation in the Golgi. The CaS receptor requires co-expression of RAMPs 1 or 3 (but not RAMP2) for trafficking to the cell surface [10]. The mechanism of action seems similar to that for the CLR, in that the RAMPs deliver the receptor from the endoplasmic reticulum to

Consequences of changes in RAMP expression/activity

There is now abundant evidence for regulation of RAMP expression, be this in disease, in response to drugs/hormones or physiologically 34, 35. Many of these studies, including those reporting RAMP regulation in heart failure, hypertension and renal failure have been reviewed [11]. More recently, knockout mouse models for each RAMP gene have been developed, allowing new insight into the function of these proteins. For example, RAMP2 and RAMP3 knockout mice have revealed distinct roles for these

The potential of RAMPs as drugs targets

In principle, RAMPs can be utilised as drug targets either directly themselves, or the complex between the RAMP and its target receptor could be targeted. A drug acting at a RAMP/receptor complex could interact with both the RAMP and the receptor. If the RAMP caused significant structural perturbation of the receptor, yielding a conformation different from that of the receptor expressed alone, then this novel structure by itself might be sufficient for selective drug binding. While most

Conclusion

While there is little solid information available as yet on how drugs can be designed to target RAMP/receptor complexes or RAMPs by themselves, there are grounds for optimism; the CGRP receptor antagonists provide proof of principle. Such agents provide novel therapeutic opportunities. New data indicate that, in addition to receptors, RAMPs may also interact with other proteins such as tubulin [59]. Although, the physiological significance of this observation for RAMP1 is currently unknown, it

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