Abstract

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of alpha-, beta-, and gamma-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the alpha-subunit. The alpha-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known alpha-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of alpha-subunit function, we performed a screen for loss-of-function mutations, using the G alpha2-subunit of Dictyostelium. G alpha2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of G alpha2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to G alpha2 function. A library of random point mutations in the g alpha2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a g alpha2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the g alpha2 insert. To date, sixteen single amino acids changes have been identified in G alpha2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the alpha-subunit. These loss-of-function mutations in the alpha-subunit Helical domain may provide important insight into its function.