G protein diversity

Citation

Strathmann, Michael Paul (1991) G protein diversity. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/wmg0-x120. https://resolver.caltech.edu/CaltechTHESIS:04112011-133018554

Abstract

The flow of information from hormone receptors through heterotrimeric G proteins to intracellular effectors constitutes a basic form of signal transduction that has been found in every eukaryotic cell examined. This mode of signaling is the basis for intercellular communication in one-celled organisms, such as S. cerevisiae, and simple eukaryotes that undergo limited development, e.g., Dictyostelium. We examined G protein diversity in the mouse to explore how G protein-mediated signal transduction has adapted to the complex signaling processes that define a multicellular organism. We found that the diversity of G protein alpha subunits is generated by a large number of distinct genes and by products of alternative splicing. Five new genes were found to encode alpha subunits that fall into two new classes. These classes, which are defined by amino acid sequence identity, have been conserved in distantly related animals; the four classes known to exist in mammals are also present in Drosophila, and three of these classes have been found in nematodes. The gene encoding Gα_o in mammals, an alpha subunit earlier characterized by biochemical means, was found to undergo alternative splicing to produce transcripts encoding two forms of the protein. In addition to the diversity among alpha subunits, we found a novel beta subunit. This was particularly surprising because biochemical evidence suggested that beta-gamma dimers are interchangeable. The possibility of combinatorial associations of alpha, beta, and gamma subunits to produce functionally distinct heterotrimers must be considered. The considerable task of analyzing gene families by cloning and sequencing necessitated the development of new techniques for these purposes. We used the polymerase chain reaction to amplify cDNA with degenerate oligonucleotide primers. The primers were designed to hybridize to regions of DNA that are conserved in all members of the gene family. In addition, we developed a technique for randomly inserting sequencing primer sites throughout a cloned region of DNA. For this purpose, we used the transposon γδ coupled with an efficient scheme for isolating and characterizing the insertions.

Thesis Files