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.
Highlights
G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors in eukaryotes, and they mediate a wide variety of cellular responses through activation of heterotrimeric G-proteins [1]
To identify peptide ligands of GPR55, the NEB C7C phage-displayed random peptide library was screened using as bait HEK293 cells that heterologously expressed HA-tagged GPR55
The GPR55 gene is abundantly expressed in lymphoid organs, such as spleen and thymus, and hyperexpressed in many human cancer cells, including B-cell multiple myeloma, lymphoma, and lymphoblastoid cells [6, 23,24,25]
Summary
G-protein-coupled receptors (GPCRs) are the largest family of membrane receptors in eukaryotes, and they mediate a wide variety of cellular responses through activation of heterotrimeric G-proteins [1]. Correlations have emerged between GPCR expression levels and tumor development [4]. LPI has pleiotropic activities in different cellular systems, and initial evidence of its role in cancer development was derived from clinical data, where LPI emerged as a marker of poor prognosis for patients with ovarian cancer [8]. This was in agreement with in-vitro studies that demonstrated high levels of lysophosphatidic acid, LPI, and their metabolites in tumor cells and transformed cells, as compared to their normal cell counterparts [7]. Rastransformed fibroblasts have a high intracellular content of LPI, which is secreted extracellularly and can stimulate cell proliferation in an autocrine manner [9]
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