Elsevier

Biochemical Pharmacology

Volume 71, Issue 3, 12 January 2006, Pages 319-337
Biochemical Pharmacology

Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library

https://doi.org/10.1016/j.bcp.2005.10.050Get rights and content

Abstract

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.

Introduction

G protein-coupled receptors (GPCRs) constitute the single largest and the most diverse family of receptors, and they are characterized with seven distinct hydrophobic regions and are known to be activated by a diverse array of extracellular ligands. In the last decade, the progress in genome sequencing has led to the discovery of new GPCR family members, and over 1000 GPCRs have been cloned from a wide range of species. Many of the novel receptors identified have no known ligand and are classified as orphan receptors. Indeed, 50% of the cloned GPCRs are orphan receptors [1]. The challenge ahead is to characterize these orphan receptors by identifying their cognate/surrogate ligands and unraveling their biological roles.

Mas oncogene was identified by a tumorigenicity assay in which NIH3T3 cells were transfected with DNA from an epidermoid carcinoma [2]. Mas was predicted to be a GPCR and has been demonstrated to be abundantly expressed in the brain and the testis [3], [4]. Activation of mas proto-oncogene results from a 5′-rearrangement while there is no nucleotide change in the coding region [2]. Similar findings were also reported in an ovarian carcinoma [5] and an acute leukaemia [6].

Phage-displayed random peptide libraries provide a good source of epitopes that could potentially function as surrogate ligands for many receptors. To construct a phage-displayed random peptide library, a pool of small random peptide epitopes are tagged to the N-terminus of M13 phage gIII coat proteins which are expressed on the phage surface [7]. Peptide sequences that have high affinity to the target receptor are commonly selected against immobilized targets by a process of repeated binding and elution cycles known as panning. The major shortcoming of using immobilized targets or fixed cells for panning is of retrieving epitopes that might not bind to the native receptor [8]. On the other hand, live cell panning facilitates the selection of functionally active ligands that recognize the native receptors [9], [10].

In the present study, we used a phage-displayed 12-mer random peptide library to identify surrogate peptides of mas that were biologically active. Initially, we enriched peptides with consensus motifs by panning a phage library against live cells that were transiently expressing mas-GFP fusion protein. A synthetic peptide MBP7 with consensus motif was identified as an agonist for mas through stimulation of mas internalization, activation of phospholipase C (PLC), and intracellular calcium mobilization in a stable cell line that over-expressed mas. To our best understanding, this is the first report identifying a peptide agonist for an orphan G protein-coupled receptor using a phage-displayed random peptide library.

Section snippets

Materials

The rabbit anti-mas receptor polyclonal antibody was raised against a putative C-terminal peptide of the mas protein (RAFKDEMQPRRQKDNC) using standard techniques as described previously [11]. SuperScript II, T4 DNA ligase, Iscove's modified DMEM medium, fetal bovine serum, penicillin/streptomycin, HT supplement, trypsin, TRIzol™ reagent, mas peptide antigen and various oligos were from Invitrogen (Carlsbad, CA, USA). The MBP7 (KAQLRRLS) peptide was synthesized in-house using Fmoc solid-phase

Characteristics of the phage-displayed random peptide library

A random peptide library displaying 12 amino acids was constructed by cloning the PCR amplified oligo templates upstream and inframe to the M13 phage coat protein gIII. The resultant library comprised more than 2.2 × 109 recombinants and 30 clones were randomly selected for analysis. Analysis of the nucleotide sequences of the 30 inserts showed that the random peptide library was predominantly in accordance with the NNS pattern and there was no bias towards any particular nucleotides (Fig. 1A).

Discussion

Mas oncogene was identified more than a decade ago and was reported to be expressed in abundance in the brain and testis. However, understanding its physiological roles has been severely hampered due to lack of information regarding its cognate/surrogate ligands. In order to obtain a pharmacological/surrogate ligand, we used a phage library displaying linear random peptide epitopes (11–16 amino acids) to identify surrogate peptide ligands for this orphan receptor. Alignment of the peptide

Acknowledgements

Special thanks to Dr. David R. Poyner for his critical comments and helpful suggestions on the project. The authors also would like to thank the excellent technical help of Kevin B.S. Chow, Helen S.N. Tsai, Denis T.M. Ip, and Wallace Yip. This work was supported in part by an RGC Direct Grant (2330-140) to WTC.

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