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Title: Studies on the rat hepatic glucagon receptor
Author: Collett, Gavin Patrick
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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Treatment of liver plasma membranes with CHAPS was shown to yield glucagon receptor in a soluble, active form. Characterisation of the CHAPS- solubilised receptor using sucrose density gradient centrifugation and gel filtration on Sepharose CL-6B showed that it exists predominantly in a highly aggregated form, and that this aggregation is specific and unchanged by alterations to the detergent concentration or ionic strength. The smallest molecular weight species for the receptor in CHAPS was determined to be ~190 kDa and this is proposed to be a trimer of the hormone binding subunit. Isolation of the active, CHAPS-solubilised receptor was attempted using affinity chromatography with immobilised glucagon, prepared by coupling bromoacetylated BioGel P-150 to the met-25 residue of glucagon. Specific binding of the receptor to the affinity gel was demonstrated. However, various elution methods (with glucagon, urea or low pH buffers) did not yield active receptor. Attempts to elute the receptor with SDS followed by characterisation on SDS- PAGE were unsuccessful due to background non-specific binding to the affinity matrix of the majority of proteins present in the CHAPS extract. Thermostability studies on the CHAPS-solubilised receptor, showing biphasic decay at 35°C, provided further evidence of possible receptor heterogeneity. It has been proposed that the desensitisation of adenylate cyclase after exposure of hepatocytes to glucagon, or other ligands which stimulate inositol phospholipid metabolism, arises as a result of phosphorylation of the glucagon receptor by protein kinase C. This was investigated by carrying out isoelectric focusing of samples of partially purified (by wheat germ agglutinin-agarose chromatography) receptor to which 125I-glucagon had been directly cross-linked by UV irradiation, using immobilised pH gradient gels and agarose IEF gels. In both cases the procedure was optimised to allow good resolution of the components of solubilised liver membranes, but focusing of the labelled receptor was not possible, presumably due to aggregation and/or precipitation at the site of sample application. An alternative approach was to look for a difference in the thermostability of the receptor in membrane preparations from control and desensitised hepatocytes. No difference was observed, even in the presence of phosphatase inhibitors, indicating either that desensitisation is not brought about by receptor phosphorylation, or that phosphorylation does occur but does not result in a change in the heat stability of the receptor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available