Abstract
BackgroundG protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression.MethodsFive deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots.ResultsInvestigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression.ConclusionWe characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.
Highlights
G protein-coupled receptor kinases (GRKs) are a family of seven serine-threonine kinases, allocated to three subfamilies, which interact with specific G protein-coupled receptors (GPCR) as well as with downstream signalling pathways [1]
Functionality of the cAMP responsive element (CRE) binding protein (CREB) binding site was verified in electrophoretic mobility shift assays (EMSA) blots
Stimulation with a protein kinase C (PKC) activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression
Summary
G protein-coupled receptor kinases (GRKs) are a family of seven serine-threonine kinases, allocated to three subfamilies, which interact with specific G protein-coupled receptors (GPCR) as well as with downstream signalling pathways [1]. GRK6 is part of the GRK4 subfamily and is expressed in a broad variety of tissues [2]. It undergoes post-translational palmitoylation (reviewed in [1]) and its expression is detectable in membrane and cytosolic subcellular fractions [3]. G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. The aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression
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