Role of G Protein‐Coupled Receptor Kinase 2 in TNFα Signaling in Colon Epithelial Cells

Abstract

G protein-coupled receptor kinases (GRKs) compose a 7-member family of serine/threonine protein kinases that are critical in the regulation of GPCR lifecycle. In addition to GPCR phosphorylation, GRKs have been shown to have a large interactome and can play a critical role in cell signaling pathways as well as in consequent cell physiological processes. We wanted to test the hypothesis that GRK2 plays an essential role in colon epithelial cell biology in response to TNFα. For this we used colon epithelial cell line (SW480) that were transfected with either control or GRK2 siRNA. The cells were treated with TNFα and assessed for various cell physiological processes including wound healing, proliferation, apoptosis, and gene expression. Our results demonstrate that GRK2 has a positive role basally in the rate of wound closure although the addition of TNFα restored that rate in the siGRK2 group to control levels. Interestingly, upon examining the levels of various gene transcripts, we observed that matrix metalloproteinases (MMP) 7 and 9 as well as urokinase plasminogen activator (uPa) (proteins involved in cell migration) were markedly upregulated in GRK2 knockdown cells. To assess the mechanism by which GRK2 affects these physiological processes, we examined MAPK and NFkB pathways following TNFα treatment. We found that knockdown of GRK2 significantly inhibits TNFa-induced IkBa phosphorylation whereas ERK2 phosphorylation was significantly enhanced. These data suggest that GRK2 may play a critical role in cellular migration and/or wound healing by modulating MMP7, 9 and uPA levels potentially via differential regulation of MAPK and NFkB signaling pathways. Together, our results provide a novel role for GRK2 in TNFa signaling in colon epithelial cells. Supported by NIH Grant.