Chapter 3 - Receptor Activity Modifying Proteins and Their Potential as Drug Targets

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Abstract

The receptor activity modifying proteins (RAMPs) are a family of membrane proteins that interact with some G protein-coupled receptors (GPCRs) to regulate their function. RAMPs can alter GPCR pharmacology and can regulate their signaling as well as trafficking to and from the cell surface. GPCRs have been successfully exploited as drug targets for many years. RAMPs therefore provide a new avenue for drug development, offering opportunities for regulating the function of therapeutically relevant RAMP-interacting GPCRs. RAMPs could be directly targeted themselves or advantage could be taken of the unique RAMP/GPCR interfaces for generating more selective drugs.

Introduction

Receptor activity modifying proteins (RAMPs) are single transmembrane spanning proteins that can associate with membrane proteins, specifically G protein-coupled receptors (GPCRs), potentially altering GPCR trafficking, pharmacology and/or signaling capabilities.1 GPCRs constitute one of the largest protein superfamilies in mammals, are estimated to comprise 2–3% of the human genome, and regulate many physiological processes.2 As a result, nearly 40% of all existing medications regulate GPCR function. Thus, these receptors are of enormous interest to the pharmaceutical industry.3 Classifications based on sequence homology and ligand structure have identified several GPCR subfamilies.4 To date, Family A is the largest family and includes receptors for neurotransmitters and hormones; Family B contains hormone receptors for large peptides, such as calcitonin (CT), glucagon and secretin; and Family C incorporates receptors for small molecules such as glutamate, GABA and calcium. Increasing evidence suggests that GPCRs can exist as homo- and/or heterodimers, or as a part of larger oligomeric complexes that can influence many aspects of GPCR activity and consequently cell function.5 In addition, they can interact with a diverse range of other proteins such as RAMPs, that can also regulate GPCR function. The association of GPCRs with RAMPs creates novel prospects for the development of highly selective ligands by providing an opportunity to achieve a higher degree of selectivity than that of drugs that target the GPCR by itself.

Section snippets

RAMPs and Their Discovery

The receptors for calcitonin gene-related peptide (CGRP), amylin (AMY) and adrenomedullin (AM) were believed to be individual GPCRs for many years; however, despite numerous attempts, efforts to clone these receptors proved difficult. In the early 1990s, an orphan GPCR was cloned from rat and human species that contained approximately 50% sequence identity to the calcitonin receptor (CTR) and was therefore named the calcitonin receptor-like receptor6., 7. (CRLR; this is now abbreviated to CLR8

RAMPs and Their Interaction with GPCRs

RAMPs have been demonstrated to interact with a number of GPCRs and their interactions with the receptors for the calcitonin (CT) family of peptides are as yet the best characterized. In addition to interacting with the CLR, RAMPs 1, 2 and 3 were also shown to interact with the CT receptor (CTR) to yield functional AMY receptors, with each CT/RAMP complex displaying a distinct phenotype.

RAMPs are generally poorly expressed at the cell surface in the absence of an interacting partner protein. In

RAMP Modulation of GPCR Pharmacology

The most well-studied consequence of RAMP interaction with GPCRs is its ability to alter the affinity and efficacy of ligands acting at the CLR and CTR, thus modulating receptor specificity. CLR by itself is not able to bind any known ligand; however, the CLR/RAMP1 complex forms a high-affinity CGRP receptor, whereas the presence of RAMP2 or RAMP3 in association with CLR gives rise to two distinct subtypes of the AM receptor, AM1 (CLR/RAMP2), and AM2 (CLR/RAMP3).8 The AM1 receptor displays a

RAMPs as Receptor Chaperones

As previously mentioned, the association of RAMP with the CLR is required for its efficient transport from the endoplasmic reticulum (ER) to the cell surface. This retention in the cell is also true for RAMPs (particularly RAMP1) when they are not associated with a receptor partner.9 In the absence of a receptor partner, RAMP1 is retained in the ER and exists as a homodimer. Interaction with a partner protein decreases the quantity of RAMP in the homodimeric form and the hetero-oligomeric form

RAMPs and Receptor Internalization

Significant evidence exists for a role of RAMPs in modulating receptor trafficking following internalization. To date, these studies have only explored the role of RAMPs interacting with the CLR, and the extent to which other identified receptor/RAMP pairings affect internalization and trafficking of the receptor is unknown. Initial evidence suggested that RAMP/CLR complexes were internalized together following agonist stimulation and that most likely, they remain complexed together throughout

RAMPs and Receptor Signaling

In addition to the role of RAMPs in modifying receptor behavior, there is also increasing evidence that RAMPs may alter the signaling phenotype of the respective GPCR. The C-terminal tail of the RAMPs has been shown to play a significant role in modulating the G protein-coupling specificity of AMY receptors. Removal of the C-terminus of RAMP1 and its exchange for the C-terminus of RAMP2 reduced the potency of CGRP in stimulating cAMP formation at the AMY1 receptor.26 In addition, AMY binding

Posttranslational Modifications of RAMPs—Glycosylation

As previously mentioned, RAMP1 lacks glycosylation sites and is unable to reach the cell surface in the absence of an interacting partner. Both RAMP2 and RAMP3 are glycosylated and in vitro assays have suggested that these RAMPs can reach the plasma membrane to some extent without an accompanying receptor.10., 27., 34. As these types of experiments rely on tagged proteins, it is unclear to what extent RAMPs 2 and 3 traffic independently in vivo. However, it is known that hetero-oligomerization

Correlation of RAMPs with Receptors In Vivo

While profiling interaction of RAMPs and their associated receptors in heterologous in vitro expression systems is informative, it is desirable to understand the contribution of the relevant RAMP/receptor pairings in vivo. This is inherently difficult, given the complexity that may arise when several RAMP and receptor partners are expressed in the same tissue. Resolving the presence of RAMPs and receptors at the cellular level is crucial to determining the important physiological interactions.

Regulation of RAMPs in Disease

Given that RAMPs form a key part of the CGRP, AM, and AMY receptors, it is predictable that changes in RAMP expression would influence the expression of these receptors and hence the sensitivity of cells and tissues to CGRP, AM, and AMY, in addition to any drugs that may be targeted to a RAMP/receptor complex. Changes in receptor activity have indeed been reported as a consequence of altering RAMP expression levels. The relatively recent development of knockout mouse models for each of the

RAMPs as Drug Targets

In principle, RAMPs could be utilized as potential drug targets to control and manage diseases such as those mentioned above and potentially others for which the role of RAMPs has not yet been understood. RAMPs could be targeted either directly themselves or by targeting the complex formed between RAMP and its interacting receptor. At this stage, due to limited structural data, structure-based drug design for RAMPs may be difficult; however, with the growing body of experimental evidence and

Concluding Remarks

Many aspects of RAMP function remain poorly understood and the full extent of their action remains to be explored. The challenge now is to gain a greater understanding of their function, their properties and the relevance of their interaction with other proteins, particularly outside cultured cell systems. These unique receptor complexes are promising drug targets and this potential needs to be explored for development of the selective pharmaceutical intervention that these RAMP complexes are

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      Thus, the RAMPs profoundly alter the behavior of CLR and CTR. Although RAMPs are best characterized for their effects on CLR/CTR, they also interact with several other class B GPCRs and with certain class A/Rhodopsin and class C/Glutamate family GPCRs, making it particularly important to understand the molecular basis for RAMP actions (Bouschet et al., 2005; Lenhart et al., 2013; Wootten et al., 2010). RAMPs provide an excellent opportunity to explore how accessory membrane proteins can modulate GPCR pharmacology.

    • Blocking metabotropic glutamate receptor subtype 7 (mGlu7) via the venus flytrap domain (VFTD) inhibits amygdala plasticity, stress, and anxiety-related behavior

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      At the present time, it remains unclear why these differences in apparent potency exist. One possibility is that endogenous proteins, such as receptor activity-modifying proteins, interact with mGlu7 in neurons conferring higher affinity for the antagonist than is measured in recombinant systems where such proteins may be absent (57). Alternatively, mGlu7 may influence synaptic plasticity in the amygdala via signaling pathway(s) distinct from those of Gi used in the recombinant assays, and the apparent potency of XAP044 for these two pathways may differ.

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      AM receptors consist of an oligomeric complex between the GPCR calcitonin receptor-like receptor (gene: Calcrl, protein: CLR) and members of the receptor activity modifying protein (RAMP) family of chaperone proteins [23–24]. Currently, the RAMP family consists of three proteins (RAMPs 1–3) sharing a similar molecular mass (160 amino acids) and structure, but differing in sequence homology, tissue distribution, and requirement for normal embryonic development [25–27]. Any of the RAMPs can pair with CLR in the endoplasmic reticulum and transport it to the plasma membrane.

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      In addition to allow the trafficking of CLR, RAMP1 and RAMP3 associate with the calcium sensing CaS receptor, facilitating its surface expression (Bouschet et al., 2005). RAMPs interact with other GPCRs without affecting receptor surface expression but rather modulating ligand specificity or receptor internalization (reviewed in Wootten et al., 2010). Receptor transport proteins RTP1 and RTP2 and the receptor expression-enhancing protein REEP1 have been identified as necessary accessory proteins for functional cell surface expression of mammalian olfactory receptors (Saito et al., 2004).

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