Balwinder Sambi

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.

Assessment of agonism at G-protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells.

Several different molecular species of phosphatidic acid (PA) bind to a G‐protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen‐activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from Gi/o). The de‐acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an ∼100 – 1000 fold lower potency compared with the different molecular species of PA. RT – PCR using gene‐specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.

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.