Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Gen Comp Endocrinol
2023 Jul 01;338:114278. doi: 10.1016/j.ygcen.2023.114278.
Show Gene links
Show Anatomy links
Trends in the evolution of the elasmobranch melanocortin-2 receptor: Insights from structure/function studies on the activation of whale shark Mc2r.
Hoglin BE
,
Miner MV
,
Erbenebayar U
,
Shaughnessy CA
,
Dores RM
.
???displayArticle.abstract???
To understand the mechanism for activation of the melanocortin-2 receptor (Mc2r) of the elasmobranch, Rhincodon typus (whale shark; ws), wsmc2r was co-expressed with wsmrap1 in CHO cells, and the transfected cells were stimulated with alanine-substituted analogs of ACTH(1-24) at the "message" motif (H6F7R8W9) and the "address" motif (K15K16R17R18P19). Complete alanine substitution of the H6F7R8W9 motif blocked activation, whereas single alanine substitution at this motif indicated the following hierarchy of position importance for activation: W9 > R8, and substitution at F7 and H6 had no effect on activation. The same analysis was done on a representative bony vertebrate Mc2r ortholog (Amia calva; bowfin; bf) and the order of position importance for activation was W9 > R8 = F7, (alanine substitution at H6 was negligible). Complete alanine substitution at the K15K16R17R18P19 motif resulted in distinct outcomes for wsMc2r and bfMc2r. For bfMc2r, this analog blocked activation-an outcome typical for bony vertebrate Mc2r orthologs. For wsMc2r, this analog resulted in a shift in sensitivity to stimulation of the analog as compared to ACTH(1-24) by two orders of magnitude, but the dose response curve did reach saturation. To evaluate whether the EC2 domain of wsMc2r plays a role in activation, a chimeric wsMc2r was made in which the EC2 domain was replaced with the EC2 domain from a melanocortin receptor that does not interact with Mrap1 (i.e., Xenopus tropicalis Mc1r). This substitution did not negatively impact the activation of the chimeric receptor. In addition, alanine substitution at a putative activation motif in the N-terminal of wsMrap1 did not affect the sensitivity of wsMc2r to stimulation by ACTH(1-24). Collectively, these observations suggest that wsMc2r may only have a HFRW binding site for melanocortin-related ligand which would explain how wsMc2r could be activated by either ACTH or MSH-sized ligands.
Fig. 1. Stimulation of wsMc2r and bfMc2r with alanine-substituted analogs of hACTH(1–24). CHO cells were co-transfected with a receptor cDNA, species specific mrap1 cDNA and a cre/luciferase cAMP reporter cDNA as described in Methods. After 48 h in culture the transfected cells were stimulated with either wild-type hACTH(1–24) or alanine-substituted analogs of hACTH(1–24) (see Supplementary Fig. 2) at concentrations varying from 10-12 M to 10-6 M. Data are presented as mean ± SEM and n = 3. The EC50 values for all the dose response curves are presented in Table 1. The transfections presented in Fig. 1A and 1B were done in the same experiment and the dose response curve for hACTH(1–24) is shown in both panels for ease in viewing. A) Stimulation of wsMc2r with either hACTH(1–24), the HFRW/AAAA analog, or the AFRW analog. B) Stimulation of wsMc2r with either hACTH(1–24), the HARW analog, the HFAW analog, or the HFRA analog. C) Stimulation of wsMc2r with hACTH(1–24), the AARRRP analog, the KKAAA analog or the KKRRP/AAAAA analog. D) Stimulation of bfMc2r with hACTH(1–24), the AFRW analog, the HARW analog, or the HFAW analog. E) Stimulation of bfMc2r with hACTH(1–24), the HFRA analog or the HFRW/AAAA analog. F) Stimulation of bfMc2r with hACTH(1–24), the AARRRP analog, the KKAAA analog or the KKRRP/AAAAA analog.
Fig. 2. Testing the activation of a wsMc2r/EC2 xtMc1r chimeric receptor. A) The amino acid sequences of wsMc2r and xtMc1r were aligned and the residues in Extracellular Domain (EC2) are highlighted in red. As described in METHODS, the EC2 domain of wsMc2r was replaced with the EC2 domain of xtMc1r. The nucleotide sequence of the wsmc2r/ec2 xtmc1r chimeric receptor cDNA is presented in Supplementary Fig. 3. B) The dose response curves for the wild-type wsMc2r and the chimeric receptor, wsMc2r/EC2 xtMc1r, both co-expressed with wsMrap1 are compared following stimulation with srACTH(1–24) as described in METHOD. The data are presented as mean ± S.E.M and n = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Evaluation of an alanine-substituted Analog of wsMrap1. A) The N-terminal and transmembrane domains of whale shark (wsMrap1), bowfin (bfMrap1) and mouse (mmMrap1) were aligned and primary sequence identity (highlighted blue) and primary sequence similarity (highlighted gray) were determined using BLOSUM (https://www.ncbi.nlm.nih.gov/Class/FieldGuide/BLOSUM62.txt). Abbreviations: δDYδ – consensus activation motif for a bony vertebrate Mrap1 ortholog. RT – reverse topology motif. TM – transmembrane domain. B) A comparison of the dose response curves for wsmc2r co-expressed with wsmrap1 and wsmc2r co-expressed with bfmrap1 following stimulation with srACTH(1–24). The data are presented as mean ± S.E.M and n = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Amino Acid sequence Alignment of Cartilaginous Fish Mc2r Orthologs. The deduced amino acid sequences of whale shark Mc2r (ws; accession number: XM_020525249.1), stingray (sr; accession number: BAU98230) and elephant shark (es; accession number: AAVX01069419.1) were aligned by inserting the minimal number of gaps, and primary sequence identity (highlighted in dark blue) and primary sequence similarity (highlighted in gray) were determined using BLOSUM https://www.ncbi.nlm.nih.gov/Class/FieldGuide/BLOSUM62.txt). The primary sequence identity/similarity of each domain is: N-terminal (15%), TM1 (91%), IC1 (71%), TM2 (91%), EC1 (42%), TM3 (84%), IC2 (95%), TM4 (68%), EC2 (33%), TM5 (61%), IC3 (46%), TM6 (61%), EC3 (93%), TM7 (88%), and C-terminal (37%). Abbreviations: TM -transmembrane domain; IC – intracellular loop; EC (extracellular loop). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Alignment of the TM4/EC2/TM5/IC3 domains of two Elasmobranch Mc2r Orthologs. The TM4/EC2/TM5/IC3 of wsMc2r and srMc2r were analyzed for primary sequence identity and similarity as described in the legend to Fig. 4. Positions that are identical are highlighted in dark blue and positions that are similar are highlighted in gray. A comparison of the percent sequence identity/similarity for just wsMc2r and srMc2r, and wsMc2r, srMC2r, and esMc2r (see Fig. 4) is presented. The TM4/EC2/TM5/IC3 precent sequence identify/similarity are highlighted with the dashed red box. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Supplementary Figure 1: Amino acids sequences of Mammalian melanocortin peptides. The cartoon represents a hypothetical POMC precursor protein with all possible melanocortin peptides positioned in the precursor protein. The amino acid sequences of bovine ACTH, αMSH, βMSH, and γMSH are presented (Nakanishi et al., 1979). The deduced sequence of δMSH is from the whale shark POMC sequence (XP_020377873).
Supplementary Figure 2: Alanine-substituted analogs of hACTH(1-24). The amino acid sequence of hACTH(1-24) is presented. The HFRW motif (message motif) is highlighted in blue, and the KKRRP (address motif) is highlighted green. Alanine substitutions for each analog are highlighted in red.
Supplementary Figure 3: A comparison of wsMc2r stimulated with either hACTH(1-24) or srACTH(1-24). The wsmc2r cDNA was co-transfected with wsmrap1 and stimulated with either hACTH(1-24) of the cartilaginous fish srACTH(1-24) at concentrations from 10-12M to 10-6M. The efficacy of the two ACTH ligands was evaluated in the CRE-Luciferase cAMP reporter assay as described in METHODS. (n =3). The EC50 values for the two ligands are presented in METHODS (Section 2b).
Supplementary Figure 4: Nucleotide sequence of chimeric receptor wsMc2r/EC2 xtMc1r. This figure presented the nucleotide sequence of wsmc2r (black letters unbolded) and the insert of the nucleotide sequence for xtmc1r Extracellular loop 2.
