Structural insights on melanocortin 5 receptor targeting to cell surface A. R. Rodrigues*, H. Almeida* and A. M. Gouveia* , ** * Laboratório de Biologia Celular e Molecular, Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto and IBMC - Instituto de Biologia Molecular e Celular, Rua do Campo Alegre 823, 4150-180 Porto, Portugal ** Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal agouveia@med.up.pt The melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) with a typical seven- transmembrane-domain structure. It is assumed that all GPCRs undergo several post-transcriptional modifications during synthesis and folding until finally target to the cell membrane [1]. The available information about the specific domains responsible for the correct targeting of the melanocortin receptors to the cell surface is scarce. Regarding MC5R, it was shown that the first 20 aminoacids of the human receptor can be deleted without affecting the ligand binding affinity, but further deletions of the N terminus resulted in total loss of binding [2]. These results were not directly correlated with the cell surface expression levels of the receptor. Thus, we aim to define the specific MC5R domains important to its correct trafficking and signaling, and preliminary results are here presented. The expression of the rat MC5R fused to the green fluorescent protein (GFP) was achieved in the HEK 293 cell line. We recently showed a correct targeting to cell surface and the attaining of a functional receptor when the GFP tag was located at the MC5R C-terminal (MC5R-GFP) [3]. In contrast, the MC5R construct containing the GFP at the N-terminus (GFP-MC5R) displays lower expression levels and was unable to target to the membrane efficiently (Fig.1). Since the attachment of a 27 kDa GFP polypeptide to the receptor N-terminus could impair its correct folding and the consequent membrane insertion, we therefore generated constructs of c-myc (8 kDa) tagged at the N- and C-terminal MC5R. Once more, MC5R-c-myc was expressed on the cell surface but c-myc- MC5R was strongly retained inside the cell (Fig. 2A). To further investigate whether the inability to traffic to the plasma membrane was cell-related, we use HeLa cells, transiently expressing the N- and C-terminal-c-myc-tagged MC5R constructs, and a similar expression pattern was observed (Fig. 2B). Our results highlight a crucial role for the N-terminus domain on MC5R membrane targeting and expression, in opposition to the findings that short N-terminal epitope tagging of MC1R, MC2R, or MC4R does not affect receptor trafficking or function [4]. Further experiments will be necessary to clarify the precise aminoacids from the N-terminus of the receptor MC5R that are fundamental to the receptor cell-surface expression. References [1] C. Dong, C.M. Filipeanu, M.T. Duvernay, G. Wu, Biochim Biophys Acta, 1768(4) (2007) 853. [2] H.B. Schiöth, S. Petersson, R. Muceniece, M. Szardenings, J.E.S. Wikberg, FEBS Lett, 410(2-3) (1997) 223. [3] A.R. Rodrigues, D. Pignatelli, H. Almeida, A.M. Gouveia, Mol Cell Endocrinol, 303 (2009) 74. [4] S. Roy, M. Rached, N. Gallo-Payet, Mol Endocrinol, 21(7) (2007) 1656. [5] A.M. Gouveia and A.R. Rodrigues are supported by POCI 2010, FSE and “Fundação para a Ciência e Tecnologia” (SFRH/BPD/34280/2006 and SFRH/BD/41024/2007). Microsc Microanal 15 (supp 3), 2009 3 Copyright 2009, LASPM doi:10.1017/S143192760999047X https://doi.org/10.1017/S143192760999047X Published online by Cambridge University Press