ACTION OF HORMONES ON ADENYLYL CYCLASE SYSTEMS

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Heterotrimeric G proteins (subunit composition alpha.Beta/gamma) mediate the effects of a large family of receptors by virtue of their activating or deactivating effects on effector systems. The last years have seen the elucidation of the primary functions of 14 out of the 16 G protein alpha subunits that are now known, as well as for Beta/gamma dimers that form as a result of G proteins activation by GTP under the receptor influence. Recent studies have shown involvement of trimeric G proteins not only in regulation of adenylyl cyclases (AC's), phospholipases (PL's), phosphodiesterases and ion channels, but also in processes such as vesicle budding from endoplasmic reticulum and Golgi networks, and in endosome acidification. Broader roles that mere signal transduction are indicated for G proteins. Mutations of the Galpha subunits that cause constitutive activation have been found in neoplasms and tumors and it has been hypothesized that some alpha subunits may be proto-oncogenes. The molecular cloning and ensuing elucidation of the predicted primary structure of AC led to the discovery of a large degree of molecular and functional diversity (8 genes with products sub-classifiable on the basis of response to Ca2+, calmodulin, Beta/gamma, and possibly activated Gi/alpha's). Up to 5 AC subtypes may be expressed in a single cell as seen with a pituitary tumor cell widely used to study regulation of adenylyl cyclase, phospholipase, ion channel and hormone secretion. During this last term we established in out laboratory the technology required to inactivate or mutate genes in embryonic stem cells and generate via injection into blastocysts animals with an altered genome. Applying this technique to knock out the alpha subunit of Gi2 we gained new information not only about its suspected role in mediating hormonal inhibition of adenylyl cyclase, but also discovered that mice deficient in alphai2 have abnormal T cell-development and function and develop an inflammatory bowel disease resembling ulcerative colitis. Whether these phenomena are causally related is not known. Research proposed in this application focusses wholly on generating novel mouse strains in which genes for alpha/0, alpha/i1, alpha i3 are inactivated and in which alpha/s and alpha/13 and alpha /i2 are mutationally activated. Through breeding, we will combine one or more of these mutations. Other modifications or inactivation of G protein subunits (e.g. alpha/o1 vs. alpha/o2) will be considered and performed depending on outcome and the speed of progress. It is hoped that these studies will 1. test for role(s) of G proteins in developmental processes as well as in cellular functions not yet thought of as targets of G protein regulation; 2. critically test the proto-oncogene potential of Galpha's in the background of a normal mouse; and 3. test for possible functions of alpha subunits for which no function is thus far known.