Establishing Experimental Protocols To Study Role of Bile Acids And Glucagon Receptor Signaling In Pathophysiology of Liver Disease Open Access
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Glucagon receptor (GR) is 7 transmembrane receptor having 3 extracellular loops, 3 intracellular loops, an extended N-terminus and cytoplasmic C-terminus tail. Binding of glucagon to GR and signaling through G proteins leads to activation of adenylyl cyclase and hence increased intracellular concentration of cAMP. During cholestatic liver disease, hepatic accumulation of bile acids leads to loss of responsiveness of GR to glucagon and there is a 50% decrease in cAMP formation. Our hypothesis is that bile acids activate PKC which then phosphorylates crucial amino acids in second or third intracellular loops or C terminus tail of GR leading to its desensitization. Our long term objectives involve characterization of GR expression on 2-D gels followed by Mass Spectrometry, radioligand binding assays and cAMP measurement in HEK293T cells transfected with normal or mutant GR genes and identify pathways of GR trafficking in HEK cells. Present study confirms that a cell confluency of 50-60%, plasmid DNA concentration of 6 µg was found to be optimal for achieving good transfection efficiency. Secondly, among the buffer systems tested to remove the bound ligand-Glucagon in radioligand binding assays, significant internalization was seen in HEK-GR cells and GR recycled back to the plasma membrane at 30 min after treatment with 100 nM glucagon when acetic acid buffer (5 mM in PBS, pH 5) was used. A protocol for obtaining satisfactorily pure plasma membrane fraction from HEK-GR cells has been discussed. Four different sets of modifications were tried in the protocol in order to optimize the centrifugation time and speed and the plasma membrane fraction was found to be rich in caveolin1 and was free from cytosolic and nuclear contamination. GR expression pattern was studied using these plasma membrane samples and the spot patterns obtained indicate the possibility that GR might exist with different levels of glycosylation and phosphorylation. According to this study, GR in HEK cells utilizes both clathrin and caveolin mediated pathways after treatment with 100 nM glucagon for 5 hrs as blocking these pathways with Nystatin and sucrose led to approximately 2 fold and 3 fold increase in GR expression respectively.