Regions of Melanocortin 2 (MC2) Receptor Accessory Protein Necessary for Dual Topology and MC2 Receptor Trafficking and Signaling

Patricia M. Hinkle,Julien A. Sebag

doi:10.1074/jbc.m804413200

Abstract

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29-37 conferred dual topology to RAMP3, normally in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.

Highlights

Is MRAP in a dual orientation throughout the cell? What regions of MRAP are important for dual topology? What regions are essential for MRAP functions? Here we show that MRAP forms antiparallel dimers in the endoplasmic reticulum (ER), and identify distinct regions of the molecule responsible for dual orientation, melanocortin 2 (MC2) receptor trafficking, and MC2 receptor signaling
Materials— hMC2 receptor with three NH2-terminal HA tags and RAMP3 were obtained from Missouri S&T cDNA Resource Center, RAMP1 constructs from Dr Ian Dickerson (University of Rochester, Rochester, NY), and yellow fluorescent protein (YFP)-F1 and YFP-F2 constructs from Dr Catherine Berlot (Weis Center for Research, Geisinger Clinic, Danville, PA) [13]
MRAP differs from other accessory proteins in having an extremely unusual dual topology [6]

Summary

EXPERIMENTAL PROCEDURES

Materials— hMC2 receptor with three NH2-terminal HA tags and RAMP3 were obtained from Missouri S&T cDNA Resource Center, RAMP1 constructs from Dr Ian Dickerson (University of Rochester, Rochester, NY), and YFP-F1 and YFP-F2 constructs from Dr Catherine Berlot (Weis Center for Research, Geisinger Clinic, Danville, PA) [13]. Surface Epitope Detection by Fixed Cell ELISA—To measure epitopes on the extracellular side of the plasma membrane, cells in 12-well plates were washed with PBS, fixed for 10 min with 2% paraformaldehyde, washed, blocked in 5% milk in PBS, and processed for ELISA as described [6] using 1:5000 monoclonal anti-V5, anti-FLAG, and anti-HA antibodies. Surface Epitope Immunoprecipitation and Western Blotting— Cells were washed, incubated with 1:2000 immunoprecipitating antibodies (anti-V5 and anti-FLAG) in F-12 media with 20 mM HEPES and 5% goat serum for 2 h at room temperature, washed, and lysed for 20 min at 4 °C with 0.1% N-dodecyl-␤maltoside in PBS with protease inhibitors.

MRAP Dual topology YFP fluorescence

RESULTS

DISCUSSION

Findings

Required for Dual topology