RESUMO
Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and ß-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.
Assuntos
Quinases de Receptores Acoplados a Proteína G/isolamento & purificação , Proteínas de Neoplasias/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Antineoplásicos/síntese química , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatografia/métodos , Clonagem Molecular , Desenho de Fármacos , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , SpodopteraRESUMO
G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting ß-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells.