Identification and characterization of surrogate peptide ligands for Mas, an orphan G protein-coupled receptor, using phage-displayed random peptide library

In the present study, efforts were made to identify and characterize surrogate peptide ligands for mas oncoprotein using phage-displayed random peptide library. Firstly, mas oncoprotein was tagged with the green fluorescent protein (GFP) at the C-terminus of mas. Localization of the fusion protein was visualized by confocal microscopy. Consistent with other GPCRs, mas-GFP expression was predominantly on the cell surface, indicating mas is an integral membrane protein. Secondly, for screening surrogate peptide ligands, random peptide libraries displaying 12 random amino acids were constructed by a PCR based method. Subtractive biopanning strategy with two heterologous mas expressing cell lines, a 24-hour transient mas-GFP expressing cell line and a stable mas over-expressing CHO Dhfr- cell line with their corresponding vector transfected cells as controls, were used to screen putative mas binding phage clones. The individual clones obtained after screening were analyzed for their mas binding activity by phage ELISA with stable mas expressing cells. Nucleotide sequence determination of the ELISA positive clones revealed many sequences with consensus motifs. Selective enrichment of some phage-encoded peptides was also noted. Two distinct peptide motifs (Mas BP6 and Mas BP7) obtained by aligning the sequences were of interest.; Phage-encoded peptides and synthetic peptides that are derived from the consensus motifs were used for binding studies by immunocytochemistry. Immunocytochemical studies on mas-GFP expressing stable cells with the phage clones (3p4A5, 3p5A110 and 3p5A190) and Alexa Fluor 546 labeled synthetic peptide (Mas BP7) revealed a punctate staining on the cell surface that was co-localized with the mas-GFP expression. However, the phages and the labeled peptide were completely internalized when incubated with a stable cell line (M80) that over-expresses mas without C-terminal tagging. This behavior of the mas protein suggests that GFP tagging might impair receptor internalization. On the other hand, synthetic Mas BP peptides and phage-encoded peptides also induced internalization of mas oncoprotein as evidenced by immunostaining with an anti-mas serum. Moreover, the internalized phages and the mas protein were found to be co-localized. These results clearly suggest that the isolated phage clones and derived peptides act as agonists of mas oncoprotein. (Abstract shortened by UMI.)...