Structure/Function Studies on the Activation Motif of Two Non-Mammalian Mrap1 Orthologs, and Observations on the Phylogeny of Mrap1, Including a Novel Characterization of an Mrap1 from the Chondrostean Fish, Polyodon spathula

Abstract

In derived bony vertebrates, activation of the melanocortin-2 receptor (Mc2r) by its ACTH ligand requires chaperoning by the Mc2r accessory protein (Mrap1). The N-terminal domain of the non-mammalian tetrapod MRAP1 from chicken (c; Gallus gallus) has the putative activation motif, W¹⁸D¹⁹Y²⁰I²¹, and the N-terminal domain in the neopterygian ray-finned fish Mrap1 from bowfin (bf; Amia calva) has the putative activation motif, Y¹⁸D¹⁹Y²⁰I²¹. The current study used an alanine-substitution paradigm to test the hypothesis that only the Y²⁰ position in the Mrap1 ortholog of these non-mammalian vertebrates is required for activation of the respective Mc2r ortholog. Instead, we found that for cMRAP1, single alanine-substitution resulted in a gradient of inhibition of activation (Y²⁰ >> D¹⁹ = W¹⁸ > I²¹). For bfMrap1, single alanine-substitution also resulted in a gradient of inhibition of activation (Y²⁰ >> D¹⁹ > I²¹ > Y¹⁸). This study also included an analysis of Mc2r activation in an older lineage of ray-finned fish, the paddlefish (p), Polyodon spathula (subclass Chondronstei). Currently no mrap1 gene has been found in the paddlefish genome. When pmc2r was expressed alone in our CHO cell/cAMP reporter gene assay, no activation was observed following stimulation with ACTH. However, when pmc2r was co-expressed with either cmrap1 or bfmrap1 robust dose response curves were generated. These results indicate that the formation of an Mc2r/Mrap1 heterodimer emerged early in the radiation of the bony vertebrates.

Keywords: MC2R; MRAP1; evolution; paddlefish.

Figure 1

Multiple amino acid sequence alignment of the N-terminal domain of osteichthyan Mrap1s. Mrap1 sequences of mouse (m), chicken (c), and bowfin (b) 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) accessed on 6 August 2022. The overall sequence identity for the three orthologs was 44% and the overall sequence similarity was 64%. Each Mrap1 ortholog contained a putative a δ-D-Y-δ (activation) motif [7].

Figure 2

Analysis of W¹⁸D¹⁹Y²⁰I²¹ motif in chicken MRAP1. (A) Activation of chicken (c) MC2R co-transfected with either wildtype cMRAP1 (positive control) or alanine substitution mutants of cMRAP1: cMRAP1−A¹⁸A¹⁹A²⁰A²¹, cMRAP1−W¹⁸/A, or cMRAP1−D¹⁹/A. (B) Activation of cMC2R co-transfected with either wildtype cmrap1 (positive control) or alanine substitution mutants of cMRAP1: cMRAP1−Y²⁰/A or cMRAP1−I²¹/A. Receptor activation was assessed after stimulation with hACTH(1-24). Data presented as mean ± SEM (n = 3).

Figure 3

Analysis of Y¹⁸D¹⁹Y²⁰I²¹ motif in bowfin Mrap1. (A) Activation of bowfin (bf) Mc2r co-transfected with either wildtype bfmrap1 (positive control) or alanine substitution mutants of bfmrap1: bfmrap1-a¹⁸a¹⁹a²⁰a²¹, bfmrap1−y¹⁸/a, or bfmrap1−d¹⁹/a. (B) Activation of bfMc2r co-transfected with either wildtype bfmrap1 (positive control) or alanine substitution mutants of bfmrap1: bfmrap−-y²⁰/a or bfmrap1−i²¹/a. Receptor activation was assessed after stimulation with hACTH(1-24). Data presented as mean ± SEM (n = 3).

Figure 4

Functional analysis of the paddlefish Mc2r. (A) When pMc2r was expressed alone, there was no activation of the receptor by hACTH(1-24). The positive control for this assay was bowfin (bf) Mc2r co-expressed with bfMrap1. Note the robust activation following stimulation with hACTH(1−24). (B) Analysis of pMc2r stimulation by hACTH(1-24) when co-expressed with various vertebrate Mrap1s: whale shark (ws) Mrap1, bfMrap1, or chicken (c) MRAP1. (C) Analysis of pMc2r stimulation by hACTH(1-24) when co-expressed with either bfMrap1 (positive control) or a bfMrap1-Y²⁰/A²⁰ mutant. (D) Analysis of ligand selectivity of pMc2r, wherein pMc2r was co-expressed with bfMrap1 and stimulated with either hACTH(1-24) or α-MSH. Data presented as mean ± SEM (n = 3).

Figure 5

Summary of Mc2r activation requirements in gnathostomes. Definition of terms: Gnathostomata, is bilaterally symmetrical with a vertebral column and true hinged jaws; Osteichthyes, has bony vertebral column; Sarcopterygii, all bony vertebrates with lobed fins or limbs; Actinopterygii, all bony fishes with ray fins; Tetrapoda, all vertebrates with four limbs; Chondrichthyes, gnathostomes with a cartilaginous vertebral column. Dashed line highlights that functional studies have not been performed on the Mc2r ortholog of the basal Sarcopterygian lineages, such as the lungfishes.

Figure 6

Multiple amino acid sequence alignment of vertebrate Mrap orthologs. (A) The amino acid sequence of N-terminal and transmembrane domains of mouse (m) Mrap1 (Accession No. NP_084120) chicken (c) MRAP1 (XR_001470382), bowfin (bf) Mrap1 (LOC: AMCT00016091, [17]), zebrafish (zf) Mrap1 (XP_001342923), and rainbow trout (rt) Mrap [accession number FR837908] 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) accessed on 6 August 2022. (B) The amino acid sequence of the N-terminal and transmembrane domains of elephant shark (es) Mrap1 (XM_007903550.1), whale shark (ws) Mrap1 (XP_020375601), and sea lamprey, Petromyzon marinus (pm) Mrap2 (BR000864.1) were aligned and analyzed as described above.

Figure 7

Proposed evolution of Mrap during the radiation of vertebrates. See text for explanation and discussion. Abbreviations: R—reverse topology motif in the N-terminal of Mrap; T—transmembrane domain in Mrap; R2—second chordate genome duplication event.