Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma

2021 ◽  
pp. 105890
Author(s):  
Tien L. Olson ◽  
Shangji Zhang ◽  
Dillon Labban ◽  
Emily Kaschner ◽  
Manuel Aceves ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Design ◽  
Protein Expression ◽  
G Protein ◽  
Receptor Kinase ◽  
Expression And Purification ◽  
G Protein Coupled Receptor ◽  
Structure Based Drug Design ◽  
Protein Expression And Purification ◽  
G Protein Coupled

scholarly journals Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247087
Author(s):  
Maike Stegen ◽  
Andrea Engler ◽  
Crista Ochsenfarth ◽  
Iris Manthey ◽  
Jürgen Peters ◽  
...  
Keyword(s):  
Protein Expression ◽  
Binding Site ◽  
G Protein ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Western Blots ◽  
G Protein Coupled ◽  
Creb Binding Site

Background 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. 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. Methods Five 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. Results Investigation 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. Conclusion We 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.

Abstract 156: Screening and characterization of inhibitors of G-protein coupled receptor kinase GRK6 as potential therapeutics for multiple myeloma

2012 ◽  
Author(s):  
Carly Griffin ◽  
Ratheesh Subramanian ◽  
Hassan S. Zaidi ◽  
Richard Marcellus ◽  
Babu Joseph ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
G Protein ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Potential Therapeutics
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