The G proteins are a family of proteins that couple a variety of membrane receptors to intracellular enzymes and ion channels. Individual cells contain many subtypes of G protein subunits in proportions which are characteristic of the different cell types. The overall goal of this project is to define the mechanisms which determine cellular levels of G protein subunits. At present, very little is known about how the level of G protein subunits are regulated: how rats of transcription, translation or protein degradation vary among G protein subunits, or which of these steps is most important in determining the level of a subunit. The problem can be divided into two parts: 1) How is the study-state level of mRNA related to the protein level? 2) What determines the study-state level of mRNA for a G protein subunit? We propose to begin with the first question, starting with the protein and, in future studies, working our way back to the gene.
The specific aims are: 1. To quantitate the steady-state levels of protein mRNA for alpha(o), alpha(i-2) and beta1 in rat liver, heart, cerebral cortex and in three types of cultured cells (GH(4), PC12 and cardiocytes). 2. To determine the rates of synthesis and degradation of alpha(o) and alpha(i-2) in cell lines. 3. To determine the ratio of translationally active mRNA for alpha(o), alpha(i-2) and beta1 to translationally inactive messenger ribonucleoprotein. In selected tissues or cells, we will determine the polysome profile for these mRNAs and determine the elongation rate by a polysome run-off assay. 4. To use in vitro translation to analyze the translational properties of cellular mRNA and mRNA transcribed from modified cDNA. The translational properties of mRNA and mRNPs will be compared to protein-free mRNA. The translational properties of mRNA made from cDNAs modified in the 5' untranslated region will be measured to analyze the role of the 5' end of the mRNA in regulation of translation. The ability of cells to respond to hormones, neurotransmitters and mitogens depends not only on receptors for these agonists, but also on the type and amount of G proteins they express. These studies will provide the first systematic analysis of some of the mechanisms which set the levels of G protein subunits and lay the foundation for future studies to understand how these mechanisms function in development, differentiation and cell growth.
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