The melanocortin-2 receptor accessory protein (MRAP) is a type I integral membrane protein that is required for cell surface expression of the melanocortin 2 receptor (MC2R). Mutations in the N-terminal region of this protein are associated with familial glucocorticoid deficiency type II (OMIM#607398).
Here we have investigated the expression and biochemical properties of MRAP using polyclonal antibodies to a conserved peptide sequence in the N-terminal region of MRAP. Western blot analysis of lysates and membrane preparations from Y1 mouse adrenocortical cells subjected to SDS-PAGE under reducing conditions revealed two proteins with molecular masses of ~16 kDa and ~32 kDa (predicted mass for mouse MRAP is 14.1 kDa). Post-translational modification at two potential N-glycosylation sites was investigated by treatment with N-Glysosidase F but the molecular masses of the two proteins were unaffected. Western blot analysis of rat organs/tissues also revealed a ~32 kDa band in adrenal glands and in ovarian tissue, indicating that MRAP may exist in vivo as a dimeric protein. To test this hypothesis SK-N-SH cells transiently transfected with MRAP labelled with a Flag epitope tag at the C-terminus were analysed in co-immunoprecipitation experiments using Flag- and MRAP- antibodies. Both Flag- and MRAP- antibodies immunopreciptated a protein with a mass of ~20 kDa, consistent with the expected mass for MRAP-flag monomer. However, Flag-antibodies also immunoprecipitated a protein of ~40 kDa, consistent with dimeric MRAP-flag. These data suggest that the N-terminal antigen targeted by the MRAP antibodies may be inaccessible when dimeric MRAP is in solution.
Collectively our data indicate that MRAP exists as a dimer in vivo and that dimerisation may occur in the N-terminal region of the protein. These findings are consistent with other G-protein-coupled receptor interacting proteins such as RAMPs (receptor activity modifying proteins), which also exist as dimeric proteins.
01 - 05 Apr 2006
European Society of Endocrinology