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Biomolecules
2022 Oct 04;1210:. doi: 10.3390/biom12101422.
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Human Melanocortin-2 Receptor: Identifying a Role for Residues in the TM4, EC2, and TM5 Domains in Activation and Trafficking as a Result of Co-Expression with the Accessory Protein, Mrap1 in Chinese Hamster Ovary Cells.
Davis PV
,
Shaughnessy CA
,
Dores RM
.
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Human melanocortin-2 receptor (hMC2R) co-expressed with the accessory protein mouse (m)MRAP1 in Chinese Hamster Ovary (CHO) cells has been used as a model system to investigate the activation and trafficking of hMC2R. A previous study had shown that the N-terminal domain of mMRAP1 makes contact with one of the extracellular domains of hMC2R to facilitate activation of hMC2R. A chimeric receptor paradigm was used in which the extracellular domains of hMC2R were replaced with the corresponding domains from Xenopus tropicalis MC1R, a receptor that does not interact with MRAP1, to reveal that EC2 (Extracellular domain 2) is the most likely contact site for hMC2R and mMRAP1 to facilitate activation of the receptor following an ACTH binding event. Prior to activation, mMRAP1 facilitates the trafficking of hMC2R from the ER to the plasma membrane. This process is dependent on the transmembrane domain (TM) of mMRAP1 making contact with one or more TMs of hMC2R. A single alanine substitution paradigm was used to identify residues in TM4 (i.e., I163, M165), EC2 (F167), and TM5 (F178) that play a role in the trafficking of hMC2R to the plasma membrane. These results provide further clarification of the activation mechanism for hMC2R.
Figure 1. Alignment of Amino Acid Sequences of hMC2R and xtMC1R. The hypothetical membrane topology of hMC2R was predicted using the TMHMM program (https://www.bioinformatics.dtu.dk, accessed on 18 October 2018). The amino acid sequence of xtMC1R was aligned to hMC2R by inserting two gaps. The predicted extracellular domains of hMC2R and xtMC1R are highlighted in red. Identical positions in both receptors are in bold red for residues in an extracellular domain and bold black for residues in a TM, IC, or C-terminal domain. Abbreviations: h (human), x (Xenopus tropicalis), EC (extracellular domain, IC (intracellular domain), TM (transmembrane domain).
Figure 2. cAMP Reporter Gene Assay: Analysis of hMC2R/xtMC1R Chimeric Receptors. (A) The diagram shows the relative location of the extracellular domains present in hMC2R (green) and the corresponding domains present in xtMC1R (red), and the organization of the chimeric receptors. Note the insertion of a cysteine residue in hMC2R/NT xtMC1R and hMC2R/EC3 xtMC1R. Abbreviations: C (cysteine), NT (N-terminal domain), EC1 (extracellular domain 1), EC2 (extracellular domain 2), EC3 (extracellular domain 3). (B) Dose response curves for wild-type hMC2R, hMC2R/NT xtMC1R (Chimeric NT), hMC2R/EC1 xtMC1R (Chimeric EC1), hMC2R/EC2 xtMC1R (Chimeric EC2), and hMC2R/EC3 xtMC1R (Chimeric EC3) all co-expressed with mMRAP1 in CHO cells as described in Methods, and stimulated with hACTH(1-24). (C) The EC50 value for each dose response curve is presented (n = 3) and the results of One-way ANOVA analysis of the EC50 values. A p value < 0.05 is highlighted in red.
Figure 3. Alignment of Amino Acid Sequences of TM4/EC2/TM5 Domains of Human MCRs: Identity/Similarity Analysis. (A) The amino acid sequences of the TM4/EC2/TM5 domains for hMC2R (AA067714.1), hMC1R (Q01726.2), hMC3R (AKI72215.1), hMC4R (NP_005903.2), and hMC5R (NP_005904.1) were analyzed for primary sequence identity/biochemical similarity using BLOSUM (https://www.ncbi.nlm.nih.gov/Class/FieldGuide/BLOSUM62.txt, accessed on 16 August 2022). Positions that are heighted in black for all five receptors are identical. Positions that are similar for all five receptors are highlighted in grey. (B) The same analysis was done for only hMC1r, hMC3R, hMC4R, and hMC5R, and the positions that are identical or similar but not found in hMC2R are highlighted in green.
Figure 4. Cell Surface ELISA Analysis of Single-Alanine mutants of hMC2R. The Cell Surface ELISA analysis was performed as described in Methods. The negative control was hMC2R expressed alone. The positive control was hMC2R co-expressed with mMrap1. All of the single alanine mutant forms of hMC2R were co-expressed with mMRAP1. The one-way ANOVA p values for mutants that results in a decrease in trafficking relative to the positive control are shown in red. (A) Analysis of TM4 single alanine mutants G162/A162, I163/A163, and T164/A164. (B) Analysis of TM4 single alanine mutants M165/A165, V166/A166, and I167/A167. (C) Analysis of TM5 single alanine mutants T177/A177, F178/A178, and T179/A179. (D) Analysis of TM5 single alanine mutants S180/A180, L181/A181, F182/A182, and P183/A183. (E) Analysis of EC2 single alanine mutants F168/A168 and H170/A170. (F) Alignment of the TM4/EC2/TM5 domains of human MC2R (h), Gallus gallus (chicken; c) Mc2r, and Lepisosteus osseous (gar; g) Mc2r. As described in the legend to Figure 3, amino acid positions that are identical are highlighted in black, and amino acid positions that are highlighted in grey are similar based on BLOSUM analysis. * indicates statistical decrease in trafficking relative to the positive control.
Figure 5. (A) This figure is a two-dimensional view of hMC2R based on the amino acid sequence presented in Figure 1. Amino acid positions highlighted in green are important for activation [4,6]. Amino acid positions highlighted in red play a role in trafficking (Figure 4). (B) A cartoon of mMRAP1 (red; not drawn to scale) is superimposed between TM4 and TM5. It is intended to show the orientation of mMRAP1 relative to hMC2R in the hMC2R/mMRAP1 heterodimer. (C) This cartoon depicts the predicted conformation of hMC2R with a “closed” HFRW binding site prior to an ACTH binding event. A diagram of mMRAP1 (red) is superimposed at the TM4/EC2/TM5 domain. The relative position of critical amino acid positions predicted to be involved in activation are highlighted in orange [19,20].
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