A single-format method to detect multiple G protein-coupled receptor (GPCR) signaling, especially Gα(12/13) signaling, presently has limited throughput and sensitivity. Here we report a transforming growth factor-α (TGFα) shedding assay, in which GPCR activation is measured as ectodomain shedding of a membrane-bound proform of alkaline phosphatase-tagged TGFα (AP-TGFα) and its release into conditioned medium. AP-TGFα shedding response occurred almost exclusively downstream of Gα(12/13) and Gα(q) signaling. Relying on chimeric Gα proteins and promiscuous Gα(16) protein, which can couple with Gα(s)- and Gα(i)-coupled GPCRs and induce Gα(q) signaling, we used the TGFα shedding assay to detect 104 GPCRs among 116 human GPCRs. We identified three orphan GPCRs (P2Y10, A630033H20 and GPR174) as Gα(12/13)-coupled lysophosphatidylserine receptors. Thus, the TGFα shedding assay is useful for studies of poorly characterized Gα(12/13)-coupled GPCRs and is a versatile platform for detecting GPCR activation including searching for ligands of orphan GPCRs.

This article is available online at http://www.jlr.org with plasma concentrations of 10-30 nM and several hundred nanomoles, respectively ( 3, 4 ). Both LPA and S1P have critical roles in multiple cellular events through G protein-coupled receptors (GPCRs). Six GPCRs have been identifi ed for LPA (LPA 1-6 ) and fi ve GPCRs have been identifi ed for S1P (S1P 1-5 ) ( 5 ), and nomenclature of these LysoGPs receptors has recently been proposed by Kihara et al. ( 6 ). These receptors are grouped into two classes, the Edg and P2Y families, respectively. LPA 1-3 and all fi ve of the S1P receptors, S1P [1][2][3][4][5] , are members of the Edg family, while LPA 4-6 are members of the P2Y family. In addition, LPA is an endogenous ligand for PPAR ␥ ( 7 ), and was shown to activate transient receptor potential cation channel subfamily V member 1 (TRPV1) channels leading to an infl ux of Ca 2+ ions through TRPV1 ( 8 ). Studies on gene-targeted mice and human genetic diseases have clearly shown that each receptor has specifi c roles in both physiological and pathological conditions. For example, LPA has a pivotal role in neurogenesis ( 9 ) and has also been implicated in the development of lung fi brosis ( 10 ) via LPA 1 . LPA exhibits unique roles in implantation of fertilized eggs via LPA 3 ( 11 ) and hair follicle formation via LPA 6 ( 12 ). LPA is produced by at least two pathways where multiple phospholipase activities are involved ( 3 ). Lysophospholipase D/autotaxin/NPP2 produces LPA from LysoGPs such as LPC, while phosphatidic acid (PA)-selective PLA 1 ␣ (PA-PLA 1 ␣ ) and PA-PLA 1 ␤ produce LPA from PA by their PLA 1 activities. In contrast, S1P is produced intracellularly by phosphorylation Abstract It is now accepted that lysophospholipids (LysoGPs) have a wide variety of functions as lipid mediators that are exerted through G protein-coupled receptors (GPCRs) specifi c to each lysophospholipid. While the roles of some LysoGPs, such as lysophosphatidic acid and sphingosine 1-phosphate, have been thoroughly examined, little is known about the roles of several other LysoGPs, such as lysophosphatidylserine (LysoPS), lysophosphatidylthreonine, lysophosphatidylethanolamine, lysophosphatidylinositol (LPI), and lysophosphatidylglycerol. Recently, a GPCR was found for LPI (GPR55) and three GPCRs (GPR34/LPS 1 , P2Y10/ LPS 2 , and GPR174/LPS 3 ) were found for LysoPS. In this review, we focus on these newly identifi ed GPCRs and summarize the actions of LysoPS and LPI as lipid mediators.

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA‐producing enzyme, phosphatidic acid‐selective phospholipase A1α (PA‐PLA1α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA6). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA‐induced hair follicle development. PA‐PLA1α‐deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA‐PLA1α was co‐localized with TACE, TGFα and tyrosine‐phosphorylated EGFR in the IRS. In PA‐PLA1α‐deficient hair follicles, cleaved TGFα and tyrosine‐phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA‐PLA1α enzyme induced P2Y5‐ and TACE‐mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA‐PLA1α–LPA–P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE–TGFα–EGFR pathway, thus underscoring the physiological importance of LPA‐induced EGFR transactivation.

Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator generated by hydrolysis of membrane phospholipid phosphatidylserine. Recent ligand screening of orphan G-protein-coupled receptors (GPCRs) identified two LysoPS-specific human GPCRs, namely, P2Y10 (LPS2) and GPR174 (LPS3), which, together with previously reported GPR34 (LPS1), comprise a LysoPS receptor family. Herein, we examined the structure-activity relationships of a series of synthetic LysoPS analogues toward these recently deorphanized LysoPS receptors, based on the idea that LysoPS can be regarded as consisting of distinct modules (fatty acid, glycerol, and l-serine) connected by phosphodiester and ester linkages. Starting from the endogenous ligand (1-oleoyl-LysoPS, 1), we optimized the structure of each module and the ester linkage. Accordingly, we identified some structural requirements of each module for potency and for receptor subtype selectivity. Further assembly of individually structure-optimized modules yielded a series of potent and LysoPS receptor subtype-selective agonists, particularly for P2Y10 and GPR174.

Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-α (TGFα) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors.