ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2008) 16 P529

Determination of dimerisation domains of the human melanocortin 4 receptor

Jessica Genetzky, Patrick Tarnow, Anne Rediger, Annette Grüters & Heike Biebermann

Charité Universitätsmedizin Berlin, Institute for Experimental Pediatric Endocrinology, Berlin, Germany.

The melanocortin 4 receptor (MC4R), a G protein coupled receptor, has a prominent role in hypothalamic weight regulation. The MC4R was recently shown to form receptor dimers. The cannabinoid 1 receptor (CB1R) is the nearest phylogenetic relative of MC4R but fails to interact with the MC4R in vitro. In this study we aim to investigate receptor domains that are necessary for MC4R interaction. Four different chimeric receptors of MC4R and CB1R were constructed. The generated constructs were transfected into COS-7 cells. Functional characterization included the determination of total expression, cell surface expression and dimerisation studies by a sandwich-ELISA approach as well as ligand binding and signal transduction properties. All constructs were investigated in comparison to MC4R homodimers. Exchanging of the transmembrane domain (TM) 1 and 2 (construct 1) or TM 4-7 (construct 2) resulted in a complete loss of expression. Total expression of constructs where the MC4R is exchanged with the following parts of the CB1R: TM4 alone (construct 3) or TM 3 to TM 4 (construct 4) could be determined but cell surface expression was reduced compared to the MC4R (85 and 58% respectively). No ligand binding for constructs could be determined therefore these constructs resulted in a total loss of function indicating the involvement of TM 4 and possibly TM 3 in ligand binding. Investigation of homodimerisation of construct 3 and 4 could demonstrate a strong interaction for construct 3 but no interaction of construct 4. For construct 3 also interaction of the MC4R could be observed. These findings point to a crucial role of TM3 and intracellular loop 2 for dimerisation of the human MC4R. Ongoing studies will closer narrow the domains of amino acids involved in interaction.

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