Peptide-guided targeting of GPR55 for anti-cancer therapy

Abstract

Expression of the lysophosphatidylinositol receptor GPR55 correlates with invasive potential of metastatic cells and bone metastasis formation of different types of tumors. These findings suggest a role for GPR55 signaling in cancer progression, including in lymphoproliferative diseases. Here, we screened a M13-phage-displayed random library using the bait of HEK293 cells that heterologously expressed full-length HA-GPR55. We selected a set of phagotopes that carried 7-mer insert peptides flanked by a pair of cysteine residues, which resulted in cyclized peptides. Sequencing of selected phagotopes dictated the primary structure for the synthetic FITC-labeled peptide P1, which was analyzed for binding specificity to immunoprecipitated HA-GPR55, and to endogenously expressed GPR55, using cells interfered or not for GPR55, as well as for co-localization imaging with HA-GPR55 at the membrane level. The peptide P1 stimulated GPR55 endocytosis and inhibited GPR55-dependent proliferation of EHEB and DeFew cells, two human B-lymphoblastoid cell lines. Our data support the potential therapeutic application of peptide ligands of GPR55 for targeting and inhibiting growth of neoplastic cells, which overexpress GPR55 and are dependent on GPR55 signaling for their proliferation.

Peptide targeting of the lysolipid-sensing GPR55 for osteoclastogenesis tuning

“…Prolonged LPI stimulation induces GPR55 down-regulation from the plasma membrane [38]. Time-course of LPI-induced internalisation of overexpressed ssGPR55 in HeLa cells indicated that this process started in the rst few minutes of LPI stimulation, and reached a plateau after 10 min (Fig.…”

“…Based on the sequence of peptide-P1 (CKKNSPTLC), both a scrambled peptide (Scr; KCLTSNCPK) with the same amino-acid composition as peptide-P1 but a different primary sequence, and an irrelevant peptide (Irr_P; CGGNGPGLC) that included mutations to all of the polar amino acids of peptide-P1, were designed. All of the peptides were cyclised using an intramolecular disulphide bond between the two cysteine residues [38]. The uorescent peptides were obtained by conjugation at the N-terminus with uorescein-isothiocyanate (FITC) with an aminohexanoic acid linker.…”

“…In a previous study, we succeeded in targeting GPR55 with peptides using whole-cell-based screening of a phage-displayed random library. The bait used was HEK293 cells that heterologously expressed human GPR55, with a library of cyclic peptides of seven residues that contained two anking cysteines presented by M13 phages [38]. Among these peptides seen to bind to GPR55, peptide-P1 (CKKNSPTLC) inhibited GPR55-dependent proliferation of two human B-lymphoblastoid cell lines [38].…”

“…The bait used was HEK293 cells that heterologously expressed human GPR55, with a library of cyclic peptides of seven residues that contained two anking cysteines presented by M13 phages [38]. Among these peptides seen to bind to GPR55, peptide-P1 (CKKNSPTLC) inhibited GPR55-dependent proliferation of two human B-lymphoblastoid cell lines [38]. To determine whether peptide-P1 can regulate RAW264.7 osteoclastogenesis, validation of its recognition of murine GPR55 was initially required.…”

“…To determine whether peptide-P1 can regulate RAW264.7 osteoclastogenesis, validation of its recognition of murine GPR55 was initially required. Although peptide-P1 shows speci city for the human receptor [38], human and murine GPR55 share protein sequence identity of only 75% (84% similarity; NP_005674.2 versus NP_001028462.2). For this validation, binding of uorescein-isothiocyanate (FITC)-conjugated peptide-P1…”