Loss-of-function mutations identified in the Helical domain of the G protein α-subunit, Gα2, of Dictyostelium discoideum

• Published in: Biochimica et biophysica acta. General subjects Vol. 1722; no. 3; pp. 262 - 270
• Main Authors: Gundersen, Robert E., You, Jianxin, Rauch, Steven, Farnham, Kate, McCarty, Christopher, Willis, Nicholas, Prince, Alison
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.04.2005
• Subjects: Dictyostelium; G proteins; Signal transduction; Signal transduction; Dictyostelium; G proteins
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2004.12.018

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 α-, β-, and γ-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 α-subunit. The α-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known α-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of α-subunit function, we performed a screen for loss-of-function mutations, using the Gα2-subunit of Dictyostelium. Gα2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of Gα2 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 Gα2 function. A library of random point mutations in the gα2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a gα2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the gα2 insert. To date, sixteen single amino acids changes have been identified in Gα2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the α-subunit. These loss-of-function mutations in the α-subunit Helical domain may provide important insight into its function.