Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2011) 26 OC5.3

ECE2011 Oral Communications Hormone metabolism and action (6 abstracts)

Identification of melanocortin 2 receptor structures responsible for specific membrane expression and ACTH binding specificity

D Fridmanis 1, , R Petrovska 1, , H B Schioth 1, & J Klovins 1,


1Latvian Biomedical Research and Study Center, Riga, Latvia; 2Department of Neuroscience, Uppsala University, Uppsala, Sweden.


Membrane expression of ACTH receptor MC2R is specifically limited to adrenal cells. In addition, unlike the other members of the evolutionary related MCRs that recognize different melanocortin peptides, the MC2R solely binds ACTH. We used cassette substitutions and mutagenesis of individual amino acids for systematic construction of 30 chimeric and mutant MC2R/MC4R receptors to identify the receptor structures determining the selectivity of MC2R in membrane trafficking and ACTH binding as well as interaction with MC2 receptor accessory protein (MRAP). We developed a new analysis method to quantify the localization of the recombinant receptors fused with enhanced green fluorescent protein in a cell membrane from the results of confocal fluorescent microscopy. NDP-MSH and ACTH mediated cAMP response was measured for all receptors. We have found that substitution of MC4R N-terminal part with homologues part from ACTHR significantly decreased the membrane trafficking of receptor. We have also identified the signal localized in TM3 and TM4 regions that is responsible for trapping MC2R in the endoplasmatic reticulum (ER) of the cell. Replacement of two sets of amino-acids (3aa each) in TM3 and one set of four amino-acids in TM4 with the corresponding amino acids from MC4R enabled the membrane transport of MC2R in ordinary BHK cell line without the MRAP. For control purposes all constructs were also co-expressed with MRAP protein in the same cell line. Results indicate that a specific structural conformation formed by TM2 and TM4 rather then primary aa sequence is important for ER arrest of MC2R. We have also found that the TM4 and TM5 in the MC2R are involved in MC2R binding selectivity. Bypassing of these arrest signals involves MRAP protein. In summary we have identified presence of ‘dual protection’ system that helps to avoid the membrane expression of MC2R involving N-terminal part and specific TM3–TM4 interaction.

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