Catherine M. Waters

Cell migration activated by platelet-derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1-phosphate receptor-1

We have previously identified a novel complex between the platelet‐derived growth factor (PDGF)? receptor and the sphingosine 1‐phosphate receptor‐1 (S1P1). The complex permits the utilization of active G‐protein subunits (made available by constitutively active S1P1 receptor) by the PDGF? receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. Therefore, an inverse agonist of the S1P1 receptor is predicted to reduce signal transduction from PDGF? receptor tyrosine kinase by blocking the constitutive activity of the G‐protein coupled receptor. SB649146 is a novel inverse agonist of the S1P1 receptor. First, SB649146 displaced the S1P1 receptor agonist dihydrosphingosine 1‐phosphate from membranes expressing the recombinant S1P1 receptor. Second, SB649146 reduced basal recombinant S1P1 receptor‐induced GTP?S binding and S1P‐induced GTP?S binding in membranes. Third, SB649146 blocked the S1P‐induced activation of p42/p44 MAPK in airway smooth muscle cells, a response that is mediated by the S1P1 receptor. We now report that inverse agonism of the S1P1 receptor with SB649146 reduced the endocytosis of the PDGF? receptor‐S1P1 receptor complex and the stimulation of p42/p44 MAPK and cell migration in response to PDGF. These findings are the first to report that a GPCR inverse‐agonist reduces growth factor‐induced receptor tyrosine kinase signaling, fundamentally broadening their mechanism of action. The data obtained with SB649146 also suggest that the constitutively active endogenous S1P1 receptor enhances PDGF‐induced cell migration.

Integrin signalling regulates the nuclear localization and function of the lysophosphatidic acid receptor-1 (LPA1) in mammalian cells

We show that LPA1 (lysophosphatidic acid receptor-1) is constitutively localized in the nucleus of mammalian cells. LPA1 also traffics from cell membranes to the nucleus in response to LPA (lysophosphatidic acid). Several lines of evidence suggest an important role for cell-matrix interaction in regulating the constitutive nuclear localization of LPA1. First, the RGDS peptide, which blocks cell matrix-induced integrin clustering and cytoskeletal rearrangement, reduced the number of cells containing LPA1 in the nucleus. Secondly, a higher proportion of cells contained nuclear LPA1 when adhesion on fibronectin-coated glass was compared with adherence to polylysine-coated glass. Thirdly, pre-treatment of cells with the Rho kinase inhibitor (Y27632) or the myosin light chain kinase inhibitor (ML9) reduced the number of cells containing nuclear LPA1. The addition of LPA and/or Ki16425 (which binds to LPA1) to isolated nuclei containing LPA1 induced the phosphorylation of several proteins with molecular masses of 34, 32, 14 and 11 kDa. These findings demonstrate that trafficking of LPA1 to the nucleus is influenced by cell-matrix interactions and that nuclear LPA1 may be involved in regulating intranuclear protein phosphorylation and signalling.

The inhibitory gamma subunit of the type 6 retinal cGMP phosphodiesterase functions to link c-Src and G-protein-coupled receptor kinase 2 in a signaling unit that regulates p42/p44 mitogen-activated protein kinase by epidermal growth factor.

The inhibitory γ subunit of the retinal photoreceptor type 6 cGMP phosphodiesterase (PDEγ) is phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. We report here that PDEγ is in a pre-formed complex with c-Src and that stimulation of cells with epidermal growth factor promotes the association of GRK2 with this complex. c-Src has a critical role in the stimulation of the p42/p44 mitogen-activated protein kinase cascade by epidermal growth factor, because c-Src inhibitors block the activation of this kinase by the growth factor. Mutation of Thr-62 (to Ala) in PDEγ produced a GRK2 phosphorylation-resistant mutant that was less effective in associating with GRK2 in response to epidermal growth factor and did not potentiate the stimulation of p42/p44 mitogen-activated protein kinase by this growth factor. The transcript for a short splice variant version of PDEγ lacking the Thr-62 phosphorylation site is also expressed in certain mammalian cells and, in common with the Thr-62 mutant, failed to potentiate the stimulatory effect of epidermal growth factor on p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to Ala) in PDEγ, which is a site for phosphorylation by p42/p44 mitogen-activated protein kinase, resulted in a prolonged activation of p42/p44 mitogen-activated protein kinase by epidermal growth factor, suggesting a role for this phosphorylation event in the negative feedback control of PDEγ.

Protean agonism of the lysophosphatidic acid receptor-1 with Ki16425 reduces nerve growth factor-induced neurite outgrowth in pheochromocytoma 12 cells

We report here a novel role for the constitutively active lysophosphatidic acid receptor‐1 (LPA1) receptor in providing Gβγ subunits for use by the Trk A receptor. This enhances the ability of nerve growth factor (NGF) to promote signalling and cell response. These conclusions were based on three lines of evidence. Firstly, the LPA1 receptor was co‐immunoprecipitated with the Trk A receptor from lysates, suggesting that these proteins form a complex. Secondly, Ki16425, a selective protean agonist of the LPA1 receptor, decreased constitutive basal and LPA‐induced LPA1 receptor‐stimulated GTPγS binding. Ki16425 reduced the LPA‐induced activation of p42/p44 mitogen activated protein kinase (MAPK), while acting as a weak stimulator of p42/p44 MAPK on its own, properties typical of a protean agonist. Significantly, Ki16425 also reduced the NGF‐induced stimulation of p42/p44 MAPK and inhibited NGF‐stimulated neurite outgrowth. Thirdly, the over‐expression of the C‐terminal GRK‐2 peptide, which sequesters Gβγ subunits, reduced the NGF‐induced activation of p42/p44 MAPK. In contrast, the stimulation of PC12 cells with LPA leads to a predominant Giα2‐mediated Trk A‐independent activation of p42/p44 MAPK, where Gβγ subunits play a diminished role. These findings suggest a novel role for the constitutively active LPA1 receptor in regulating NGF‐induced neuronal differentiation.

Experimental systems for studying the role of G-protein-coupled receptors in receptor tyrosine kinase signal transduction.

Early conception of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) signaling pathways was that each represented distinct and linear modules that converged on downstream targets, such as p42/p44 mitogen-activated protein kinase (MAPK). It has now become clear that this is not the case and that multiple levels of cross-talk exist between both receptor systems at early points during signaling events. In recent years, it has become apparent that transactivation of receptor tyrosine kinases by GPCR agonists is a general phenomenon that has been demonstrated for many unrelated GPCRs and receptor tyrosine kinases. In this case, GPCR/G-protein participation is upstream of the receptor tyrosine kinase. However, evidence now demonstrates that numerous growth factors use G proteins and associated signaling molecules such as beta-arrestins that participate downstream of the receptor tyrosine kinase to signal to effectors, such as p42/p44 MAPK. This article highlights experimental approaches used to investigate this novel mechanism of cross-talk between receptor tyrosine kinases and GPCRs.