Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library.
In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation. Characterization of the representative phage clones and the synthetic peptide MBP7 by immunocytochemistry revealed a strong punctate cell surface staining in CHO cells expressing mas-GFP fusion protein. The isolated phage clones and synthetic peptide MBP7 induced mas internalization in a stable CHO cell clone (MC0M80) over-expressing mas. In addition, MBP7-stimulated phospholipase C activity and intracellular calcium mobilization in these same cells. In summary, we have demonstrated a systematic approach to derive surrogate peptide ligands for orphan GPCRs. With this technique, we have identified two conserved peptide motifs which allow us to identify potential protein partners for mas, and have generated a peptide agonist MBP7 which will be invaluable for functional characterization of the mas oncogene.
Bikkavilli
Rama Kamesh
RK
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
Tsang
Sup-Yin
SY
Tang
Wai-Man
WM
Sun
Jing-Xin
JX
Ngai
Sai-Ming
SM
Lee
Susanna Sau-Tuen
SS
Ko
Wing-Hung
WH
Wise
Helen
H
Cheung
Wing-Tai
WT
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection
D007295:Inositol Phosphates; D008024:Ligands; D008565:Membrane Proteins; D019151:Peptide Library; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D002118:Calcium