The purpose of this study is to develop a detailed molecular understanding of hormonal regulation of carbohydrate metabolism of the liver. We propose to continue our studies on the glucagon stimulated adenylyl cyclase pathway. Our studies will focus on the glucagon receptor and its interaction with GTP-binding protein, G/s which is involved in transmitting the hormone signal to the catalyst of adenylyl cyclase. In this project period we propose to complete purification of the functional receptor by a four step chromatographic procedure involving (1) DEAE Sephacel chromatography (2) wheatgermlectin Sepharose chromatography (3) HPLC gel filtration and (4) glucagon agarose-affinity chromatography. The pure receptor will be reconstituted with G/s into phospholipid vesicles and the receptor dependent activation of G/s will be studied. The questions that are to be asked include (1) does the glucagon receptor promote GDP release if the G/s is loaded with GDP (2) does the glucagon receptor accelerate GTP binding if vacant G/s is used (3) what is the role of the low affinity Mg/2+ site in the activation of G/s and how does the receptor affect the Mg/2+ requirement, and (4) does the glucagon receptor provide the stratum on which there is dissociation of the GTP occupied alpha(s) subunit from the beta gamma subunits of G-proteins. We also intend to purify the glucagon receptor by a two step procedure, with high yield without concern for biological activity. The purpose is to collect sufficient amounts of receptor protein to be able to generate fragments by CNBR cleavage followed by isolation of the fragments using reverse phase HPLC. The individual peptides will be sequenced. Synthetic peptides, encoding the sequences obtained will be made and antibodies raised against these synthetic peptides. Using relevant sequences that belong to the glucagon receptor, we will construct labeled oligonucleotide probes, and screen a human liver cDNA library. Positive clones will be isolated, mapped using restriction endonucleases. The clones will be sequenced to determine if they encode the sequences that we have previously obtained from peptide sequencing. If necessary to isolate the full length cDNA primer extension followed by transformation and rescreening will be performed. The isolated clone will be expressed in xenopus oocytes, to determine if a glucagon stimulated adenylyl cyclase system can be generated upon injection of the glucagon receptor mRNA. From the full length cDNA clone, the primary sequence of the glucagon receptor will be deduced.
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