Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking

Department of Physiology, Michigan State University, East Lansing, Michigan 48824, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2005; 280(10):9297-307. DOI: 10.1074/jbc.M413786200
Source: PubMed


RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking.

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    • "Radiolabelled CGRP cross-links to proteins equivalent in size to RAMP1 alone, CLR alone and RAMP1/CLR combined suggesting that the ligand makes contact to both components of the CGRP receptor (McLatchie et al., 1998; Aldecoa et al., 2000; Hilairet et al., 2001). Also, the association of CLR with each of the RAMPs is maintained during agonist-induced receptor internalization with CLR/RAMP1 and CLR/RAMP2 being primarily targeted for degradation while CLR/RAMP3 can either be recycled or degraded depending on the cellular context (Kuwasako et al., 2000; Bomberger et al., 2005). Site-directed mutagenesis has also shown that the tryptophan residue at position 84 on RAMP1 is important for agonist potency on both the CGRP and AMY 1 receptor (Gingell et al., 2010; Moore et al., 2010). "
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    ABSTRACT: The clinical effectiveness of antagonizing the calcitonin gene-related peptide (CGRP) receptor for relief of migraine pain has been clearly demonstrated, but the road to the development of these small molecule antagonists has been daunting. The key hurdle that needed to be overcome was the CGRP receptor itself. The vast majority of the current antagonists recognize similar epitopes on the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). RAMP1 is a relatively small, single, transmembrane-spanning protein and along with the G-protein-coupled receptor CLR comprise a functional CGRP receptor. The tri-helical extracellular domain of RAMP1 plays a key role in the high affinity binding of CGRP receptor antagonists and drives their species-selective pharmacology. Over the years, a significant amount of mutagenesis data has been generated to identify specific amino acids or regions within CLR and RAMP1 that are critical to antagonist binding and has directed attention to the CLR/RAMP1 extracellular domain (ECD) complex. Recently, the crystal structure of the CGRP receptor ECD has been elucidated and not only reinforces the early mutagenesis data, but provides critical insight into the molecular mechanism of CGRP receptor antagonism. This review will highlight the drug design hurdles that must be overcome to meet the desired potency, selectivity and pharmacokinetic profile while retaining drug-like properties. Although the development of these antagonists has proved challenging, blocking the CGRP receptor may one day represent a new way to manage migraine and offer hope to migraine sufferers.
    Preview · Article · Aug 2011 · British Journal of Pharmacology
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    • "RAMP proteins may also influence calcitonin family receptor trafficking via PSD95/Disc large/Zona Occludens (PDZ) protein interactions with a PDZ domain binding motif localized at the end of the carboxyl-terminal tail of RAMP3. Specifically, RAMP3 interacts with N-ethylmaleimidesensitive factor (NSF) to regulate the agonist-stimulated recycling of the CRLR (Bomberger et al., 2005a; Kuwasako et al., 2006) and Na + /H + exchanger regulatory factor 1 (NHERF), which is involved in regulating the endocytosis of CRLR (Bomberger et al., 2005b). Thus, RAMP proteins not only regulate the pharmacology of these GPCRs but also the intracellular trafficking and post-translational modification of the receptors that are essential for the regulation of signal transduction via these receptors. "
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    ABSTRACT: GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of β-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs. LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit To view the 2010 themed section on the same topic visit
    Full-text · Article · Jun 2011 · British Journal of Pharmacology
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    • "While the N-terminus is the major determinant of receptor pharmacology (Fraser et al., 1999; Zumpe et al., 2000), the TM and C-terminus have been suggested to be important in RAMP–receptor interactions (Zumpe et al., 2000; Udawela et al., 2006b) and intracellular signalling for AMY receptors (Udawela et al., 2006a; Morfis et al., 2008) respectively . Furthermore, the C-terminus of RAMP also possesses some specific functional features including an endoplasmic reticulum retention signal in human RAMP1 and type-1 PDZ1 recognition sequence in human RAMP3, which directs CL/RAMP3 complexes to recycling (Kuwasako et al., 2000; Hilairet et al., 2001a; Steiner et al., 2002; Bomberger et al., 2005). Studies on the TM region and C-terminus of RAMPs have been reviewed elsewhere (Hay et al., 2006b; Parameswaran and Spielman, 2006; Sexton et al., 2009). "
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    ABSTRACT: The receptor activity-modifying proteins (RAMPs) are a family of three single transmembrane proteins that have been identified as accessory proteins to some G-protein-coupled receptors (GPCRs). They can regulate their pharmacology, forward trafficking and recycling, depending on the GPCR. The best characterized receptor complexes formed by RAMPs and GPCRs are the calcitonin peptide family receptors. The association of RAMP1 with the calcitonin receptor-like receptor (CL) constitutes the calcitonin gene-related peptide receptor, whereas RAMP2 or 3 with CL generates adrenomedullin receptors. In this case, the RAMPs substantially alter the pharmacology and trafficking properties of this GPCR. Amylin receptor subtypes are formed from calcitonin receptor (CTR) interactions with RAMPs. Although the RAMPs themselves are not responsive to calcitonin peptide family ligands, there is clear evidence that they participate in ligand binding, although it is still unclear whether this is by directly participating in binding or through allosteric modulation of CL or CTR. A considerable amount of mutagenesis data have now been generated on RAMPs to try and identify the residues that play a role in ligand interactions, and to also identify which residues in RAMPs interact with CL and CTR. This review will focus on RAMP mutagenesis studies with CL, summarizing and discussing the available data in association with current RAMP models and structures. The data reveal key regions in RAMPs that are important for ligand binding and receptor interactions.
    Full-text · Article · Dec 2009 · British Journal of Pharmacology
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