Davis PE , Wilkinson EC , Dores RM .

148247 Long Premed Research Fund

	Figure 1. Alignment of Human, Rainbow Trout, and Xenopus tropicalis MC2R Orthologs. The amino acid sequences of human hMC2R (accession number: AA067714.1), rainbow trout (rtMC2R; Onchorhynchus mykiss; accession number: EU119870) and Xenopus tropicalis (xtMC2R; accession number: XP002936118.1) were aligned based on primary sequence identity. The TMHMMServer, v. 2.0-DTU (http://www.cbs.dtu.dk/services/TMHMM-2.0//) was used to identify transmembrane domains (TM; underlined). The locations of the transmembrane domains (TM), intracellular loops (IC), and extracellular loops (EC) of hMC2R are labeled. Amino acid positions in human MC2R that are proposed to be in the HFRW binding site [10] are marked with a (*). The amino acid positions in hMC2R that Chen et al. [11] identified as essential for activation are highlighted in red. H170 in EC2 that Chung et al. [12] identified as essential for activation is highlighted in green. The number of positions that are identical in all three sequences is 38%.
	Figure 2. Dose response curves for xtMC2R, and single alanine mutants of xtMC2R. The wild-type receptor (xtMC2R) and single alanine mutants of xtMC2R were co-expressed with cMRAP in Chinese hamster ovary (CHO) cells as described in Materials and Methods. After two days in culture the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10−12 M to 10−6 M. The data points are mean + SEM, n = 3. The data was analyzed by one-way ANOVA as described in METHODS, and EC50 values are presented in Table 1. (A) WT xtMC2R, G154/.A, I155/A, I157/A, I158/A. (B) WT xtMC2R, M159/A, L160/A, F161/A, H162/A. (C) WT wtMC2R, D163/A, T164/A, M165/A. (D) WT xtMC2R, I167/A, I168/A, C169/A. (E) WT wtMC2R, L170/A, T171/A, V172/A. (F) XT xtMC2R, M173/A, F174/A, L175/A.
	Figure 3. Cell Surface ELISA analysis of xtMC2R mutants I158/A, F161/A, V172/A, M173/A. xtMC2R and xtMC2R mutants, I158/A, F161/A, V172/A, M173/A were separately co-transfected with cMRAP1 in CHO cells and trafficking of the receptor to the plasma membrane was evaluated by cell surface ELISA as described in Material and Methods. (A) Negative control, xtMC2R expressed alone; positive control, xtMC2R + cMRAP1, mutants analyzed: I158/A xtMC2R + cMRAP1, and F161/A xtMC2R + cMRAP1. Note that there was a significant increase in the trafficking when xtMC2R was co-expressed with cMRAP1 as compared to xtMC2R expressed alone (p < 0.001). (B) Negative control, xtMC2R expressed alone; positive control, xtMC2R + cMRAP1, mutants analyzed: V172/A xtMC2R + cMRAP1, and M173/A xtMC2R + cMRAP1. Note that there was a significant increase in the trafficking when xtMC2R was co-expressed with cMRAP1 as compared to xtMC2R expressed alone (p < 0.001). Data points are mean + SEM; n = 3.
	Figure 4. Western blot analysis - protein expression of xt MC2R single alanine mutants, I158/A, F161/A, V172/A, and M166/A. (A) This figure shows the overall protein expression of WT and single alanine mutants xMC2R I158/A, F161/A, V172/A, and M166/A transiently transfected in CHO cells with cMRAP. Whole cell lysates were prepared in in RIPA buffer and 500 µg total protein was loaded into a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (BioRad, Herculus, CA, USA) to perform western blot analysis. a-Tubulin was used as a loading control. (B) This figure shows the quantification of overall WT hMC2R or mutant expression. V5 tagged receptor expression was normalized to a-Tubulin and compared using a one-way ANOVA.
	Figure 5. Schematic representation of human, rainbow trout, and X. tropicalis MC2R. The diagrams for the proposed positioning of transmembrane domains in the MC2R orthologs for X. tropicalis, human, and rainbow trout is based on the alignments presented in Figure 1, and the presence of a disulfide bridge between cysteine residues in TM1 and TM7 that is essential for functional activation of the receptor. The transmembrane domains (TM) are represented as numbered cylinders. TMs shaded in yellow form the proposed HFRW binding site. Extracellular domains are green lines. The N-terminal domain is capped with “NH3+.” Extracellular Loop 2 is shown as a green dotted line. Intracellular domains are orange dashed lines. The C-terminal domain is capped by “COO-.” The disulfide bridge between the TM1 and TM7 [32] is a black dotted line. In each diagram, critical residues associated with the proposed HFRW binding site are a red dot. In X. tropicalis MC2R the residues that may play a role in the HFRW binding pocket are E72 (TM2), D95 (EC1) and D99 (TM3), and W230, F233, and H236 (TM6). In human MC2R the residues that play a role in the HFRW binding pocket are E80 (TM2), D103 (EC1), D107 (TM3), W233, F236, and H239 (TM6). For rainbow trout MC2R the residues that may play a role in the HFRW binding pocket are E73 (TM2), D96 (EC1), D100 (TM3), W231, F234, and H237 (TM6).

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