G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.

In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of G␣s-and G␣q-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate G␣q signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine 2C , which is coupled to G␣q. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with G␣q, direct activation of G␣q signaling (by AlF 4 Ϫ ) was desensitized by GRK2-Nter, indicating an effect at the G␣-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the G␣q subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with G␣q (only when activated) but not with G␣s and G␣o. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to G␣q and to regulate its signaling.

Evidence indicates that lipoxygenases (LO) may play a role in cancer cell survival. We show that human malignant pleural mesothelial (MM) cells, but not normal mesothelial (NM) cells, express a catalytically active 5-LO. Pharmacological or genetic inhibition of MM cell 5-LO determined nucleosome formation and induced a DNA fragmentation pattern typical of apoptosis. This was completely reversed by exogenously added 5(S)-HETE but not by 12(S)-, 15(S)-HETE, or leukotriene (LT)B4. A 5-LO antisense oligonucleotide potently and time-dependently reduced vascular endothelial growth factor (VEGF) mRNA and constitutive VEGF accumulation in the conditioned media of MM cells. When NM cells were transfected with a 5-LO cDNA, basal and arachidonic acid-induced VEGF formation increased consistently by 6- and 12-fold, respectively. This was associated with a significant increase in DNA synthesis that was counteracted by a specific anti-VEGF antibody. Arachidonic acid and 5(S)-HETE also potently stimulated the activity of a VEGF promoter construct. Thus, 5-LO is a key regulator of MM cell proliferation and survival via a VEGF-related circuit.

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