Article

Novel function for receptor activity-modifying proteins (RAMPs) in post-endocyctic receptor trafficking. J Biol Chem

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

ABSTRACT 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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    • "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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    • "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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