title: Replacement of short segments within transmembrane domains of MC2R disrupts retention signal.
abstract: The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1-24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).
authors: Latvian Biomedical Research and Study CentreRatsupites 1, LV-1067 Riga, LatviaDepartment of NeuroscienceUppsala University, BMC, PO Box 593, SE751 24 Uppsala, Sweden.
authors: Davids
authors: D
authors: Fridmanis
authors: Latvian Biomedical Research and Study CentreRatsupites 1, LV-1067 Riga, LatviaDepartment of NeuroscienceUppsala University, BMC, PO Box 593, SE751 24 Uppsala, Sweden.
authors: Ramona
authors: R
authors: Petrovska
authors: Latvian Biomedical Research and Study CentreRatsupites 1, LV-1067 Riga, LatviaDepartment of NeuroscienceUppsala University, BMC, PO Box 593, SE751 24 Uppsala, Sweden.
authors: Dace
authors: D
authors: Pjanova
authors: Latvian Biomedical Research and Study CentreRatsupites 1, LV-1067 Riga, LatviaDepartment of NeuroscienceUppsala University, BMC, PO Box 593, SE751 24 Uppsala, Sweden.
authors: Helgi B
authors: HB
authors: Schiöth
authors: Latvian Biomedical Research and Study CentreRatsupites 1, LV-1067 Riga, LatviaDepartment of NeuroscienceUppsala University, BMC, PO Box 593, SE751 24 Uppsala, Sweden klovins@biomed.lu.lv.
authors: Janis
authors: J
authors: Klovins
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D044103:Receptor, Melanocortin, Type 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction
chemical_list: D044103:Receptor, Melanocortin, Type 2; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP
keywords: ACTHR; G-protein-coupled receptor; GFP; MC2R; MRAP; chimeric receptor; melanocortin; membrane transport; rhodopsin