Structure and function of the melanocortin2 receptor accessory protein (MRAP)

Abstract

The melanocortin2 (MC2), or ACTH receptor, requires MC2 receptor accessory protein (MRAP) for function, and individuals lacking MRAP are ACTH-resistant and glucocorticoid-deficient. MRAP facilitates trafficking of the MC2 receptor to the plasma membrane and is absolutely required for ACTH binding and stimulation of cAMP. MRAP, which contains a single transmembrane domain, has a unique structure, an antiparallel homodimer. It can be isolated from the plasma membrane in a complex with the MC2 receptor. A short sequence just aminoterminal to the transmembrane domain of MRAP is essential for dual topology, while the transmembrane region is not; both are necessary for function. Deletion or alanine-substitution of other aminoterminal regions yields MRAP mutants that promote surface expression of the MC2 receptor but not receptor signaling. These results identify two distinct actions of MRAP: to permit trafficking of the MC2 receptor, and to allow surface receptor binding and signaling.

Fig. 1

Topology of endogenous MRAP. Live OS3 cells, mouse adrenocortical cells that do not express the MC2 receptor (Schimmer, 1972), were stained with affinity-purified rabbit antibodies against the N-terminal (amino acids 18-32) or C-terminal (amino acids 89-108) domains of mouse MRAP, then washed and incubated with fluorescent secondary antibody. Antibodies were added to live cells and could bind only to external epitopes. Images were deconvoluted using Metamorph software. Control experiments showed that intracellular epitopes were not stained in this protocol (Sebag and Hinkle, 2007).

Fig. 2

Top: Use of bimolecular fluorescence complementation to probe MRAP topology MRAP was fused to fragments of YFP as shown and the proteins expressed in CHO cells. Bottom: Cells were transiently transfected with MC2 receptor fused at the aminoterminus to the YFP1 fragment of YFP and MC2 receptor fused at the carboxylterminus to the YFP2 fragment. Cells were incubated with ER-tracker, which labels all cells, and imaged. As shown in Sebag and Hinkle (Sebag and Hinkle, 2008), strong fluorescence was observed in the ER and plasma membrane only when YFP fragments were present on opposite ends of the MRAP molecule.

Fig. 3

Effect of MRAP on MC2 receptor localization. Left: MC2 receptor fused at the C-terminus to tandem dimer tomato, a non-dimerizing red protein, was expressed in CHO cells with or without MRAP (Shaner et al., 2004). Right: Cells expressing MC2 receptor with or without MRAP were incubated for 20 min with vehicle or 100 nM ACTH, plus 0.1 mM isobutylmethylxanthine, and cAMP measured.

Fig. 4

Importance of MRAP regions. CHO cells expressing wildtype MC2 receptor and various MRAP and RAMP3 constructs, as shown, were tested for dual orientation at the plasma membrane, surface expression of MC2 receptor and ACTH-stimulated cAMP. Data are summarized from Sebag and Hinkle (Sebag and Hinkle, 2007; Sebag and Hinkle, 2008).

Fig. 5

Model of MRAP action. Shown is a schematic representation of the two distinct roles of MRAP: facilitating MC2 receptor translocation to the plasma membrane, and permitting MC2 receptor to bind ACTH and signal via Gs to adenylyl cyclase (AC). Black circles depict glycosylated regions. Replacement of amino acids 18-21 (LYDI) with Ala residues yields a protein that promotes receptor trafficking but not signaling. Removal of amino acids 31-37 produces a nonfunctional monomeric protein with an N_exo orientation. Replacing the transmembrane domain of MRAP with the corresponding region from RAMP3 gives a protein with dual topology that is unable to facilitate MC2 receptor trafficking or signaling.