Picture from Bowitch, Alexander et al. (2022) MicroPubl Biol

Figure 1. Human PRMT5 methylates the third intracellular loop of the C. elegans OCTR-1 receptor and the human alpha-2A adrenergic receptor (ADRA2A) in vitro.(A) Alignment showing conservation of the predicted arginine methylation motifs in the C. elegans OCTR-1 and humanADRA2A receptors. The entire third intracellular loop (3rd ICL) of OCTR-1 is predicted to have ~121 amino acids,representing residues 203-323 [SWISS-MODEL, based on (Qu et al. 2020)]; only residues 295-322 of the 3rd ICL are shownand aligned with the corresponding residues of the 3rd ICL of ADRA2A. Arginines within the predicted methylation motifs (RXR) are shown in red. (B and C) Representative blots for the in vitro methylation assays. A wild-type (WT) and mutant(RA) recombinant fragment of the 3rd ICL of OCTR-1 (residues 295-322) and ADRA2A (residues 362-387), flanked both amino- and carboxy-terminally with an S-tag to increase solubility, and fused to glutathione S-transferase (GST) were used in an in vitro methylation assay with active recombinant human PRMT5. There are no arginines within the S-tag. A fragment of the human D2 dopamine receptor was used as the positive control for methylation (Likhite et al. 2015) and GST alone served as the negative control. Top: Autoradiographs show that WT OCTR-1 and ADRA2A are methylated by PRMT5. Fragments with the conserved arginines (shown in red, Figure 1A) changed to alanines were not efficiently methylated. Monceau S staining of the polyvinylidene difluoride (PVDF) membranes was performed to demonstrate equivalent loading of the WT and RA receptor fragments. Bottom: Quantification of the degree of methylation of the receptor fragments based on densitometric analysis of the autoradiographs. Loss of the methylation motif resulted in a 67% decrease in OCTR-1 methylation and a 90% decrease in ADRA2A methylation. Data are means, +/- the standard error of the mean (SEM) from three independent experiments. *** = p < 0.001.