The G proteins are a versatile family of proteins which play a central role in transducing hormonal and sensory signals across the plasma membrane. They are, therefore, a critical point of control for a variety of agents which modulate cellular functions. Over the past 5 years, work from this and other laboratories has revealed an unexpected diversity in G protein Alpha, Beta and Y subunits. From its outset, a fundamental thesis of the work from this laboratory has been that understanding hormonal regulation of cell function will require isolating the transducing components and defining the structural features essential for their actions. Our goal is to assign functions to the components, to define the requirements for their interaction with each other, and eventually, to understand the molecular topography of the functional units. Such information may, in the future, allow development of drugs that modulate hormonal responses, and perhaps lead the ways of correcting the abnormal function of this system in diseased or malignant cells.
The first aim of the application is to identify structurally and functionally important regions of Alpha, Beta and Y subunits. We propose to clone the cDNA for the Y subunit of the hormone receptor-linked BY. This will be done in order to obtain the amino acid sequence of the Y subunit, which is essential for further chemical studies. We propose to define the functionally important regions of Alpha, Beta and Y subunits using chemical techniques. We will analyze the functional consequences of discrete chemical modifications of Alpha and Beta Y. We will determine the precise location of disulfide bonds in Alpha and Beta subunits to define which parts of the linear sequences are folded into apposition. We will map the contact regions between Alpha, Beta and Y subunits. The functional regions of the Alpha, Beta and Y subunits will also be probed by immunological studies using monoclonal antibodies and antibodies made against peptides derived from subunits expressed in E. coli. The second major specific aim is to purify and characterize Alpha z, the putative pertussis toxin-insensitive Alpha subunit. The third major specific aim is to express the G proteins in the yeast Saccharomyces cerevisiae in order to establish a system which will allow analysis of the relationship of structure to function by site-directed mutagenesis.
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