Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2

Valerie Chen,Antonio E Bruno,Laura L Britt,Ciria C Hernandez,Luis E Gimenez,Alys Peisley,Roger D Cone,Glenn L Millhauser

doi:10.1074/jbc.ra120.015482

Abstract

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein-coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein-coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.

Highlights

The melanocortin receptor accessory protein 2 (MRAP2) regulates several G-protein coupled receptors (GPCRs) that play critical roles in the regulation of energy homeostasis, and heterozygous MRAP2 variants have been identified in obese humans[1,2,3,4]
The dual topology and oligomeric state of MRAP1 is dictated by a short polybasic segment in the N-terminal domain that is directly adjacent to the transmembrane domain (Figure 1C)
These results show that the conserved polybasic segment that is required for the dual topology of MRAP1 is not required for the dual topology of MRAP2

Summary

INTRODUCTION

The melanocortin receptor accessory protein 2 (MRAP2) regulates several G-protein coupled receptors (GPCRs) that play critical roles in the regulation of energy homeostasis, and heterozygous MRAP2 variants have been identified in obese humans[1,2,3,4]. The orientation of most membrane proteins is predicted by the “positive inside rule,” where the charged amino acids flanking the transmembrane domain determine the overall orientation such that the more positive region faces the cytosol 18,19. Based on this rule, MRAP1 is predicted to insert into the membrane in both orientations, which agrees with experimental data. Despite the important role MRAP2 plays in the modulation of energy homeostasis, the sequence features within MRAP2 that dictate membrane orientation and dimerization are unknown. MRAP1’s membrane orientation and oligomeric state are dependent on a short polybasic segment adjacent to the transmembrane domain 13,14. Understanding the molecular details that determine MRAP2’s oligomeric state and membrane orientation will aid in elucidating the mechanism by which MRAP2 regulates GPCRs that are essential for metabolic processes

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES