Biochemical and molecular characterization of regulator of G protein signaling 14

Numerous signaling molecules including hormones, neurotransmitters, and cytokines activate heptahelical G protein-coupled receptors (GPCRs). GPCRs use G proteins to translate extracellular cues into intracellular signals via the generation of second messengers. Amplitude and duration of G protein responses is highly regulated. Hydrolysis of GTP by the alpha subunit of heterotrimeric G proteins results in signal termination. This process is accelerated by members of the R&barbelow;egulator of G&barbelow; protein S&barbelow;ignaling (RGS) family, which act as G&barbelow;TPase A&barbelow;ctivating P&barbelow;roteins (GAPS) for Galpha subunits. GAP activity is harbored in a protein domain of approximately 120 amino acids known as the RGS box. The focus of this dissertation is RGS14, an RGS protein highly expressed in tissues of the nervous and immune systems. In addition to an RGS domain, RGS14 contains a GoLoco domain, which acts as a G&barbelow;uanine Nucleotide D&barbelow;issociation I&barbelow;nhibitor (GDI) for Galpha subunits, and tandem Raf-like Ras/Rap binding domains that interact with the small GTPases Rap1 and Rap2.; The ability of RGS14 to interact with both monomeric and heterotrimeric G proteins led me to hypothesize that RGS14 could function as an integrator of G protein signaling. RGS14 displays the properties of a Rap effector. Only active Rap binds to RGS14 and mutations in the putative effector region abolish RGS14 interaction. Therefore, I reasoned that Rap binding to RGS14 might change RGS14's biochemical activities. To test this, I examined RGS14 activity towards heterotrimeric G proteins either as a GAP or as a GDI in the presence of Rap2A. I found that Rap2A binding had no effect on RGS14 GAP or GDI activity. I also explored the role that interdomain interactions by multiple Galpha subunits may play in regulating RGS14 function. RGS14 GAP activity towards Goalpha was unaltered by the binding of Gialpha1 to its GoLoco domain. Finally, I determined whether RGS14 was able to inhibit nucleotide exchange by Gialpha1 bound to Gbetagamma . I found that the G protein heterotrimer was resistant to RGS14 GDI activity.; In characterizing RGS14's biochemical properties, I made the discovery that RGS14 GDI activity was highly selective for the Gialpha family members Gialpha1 and Gialpha3. Previous work described the importance of residues in the GoLoco peptide and in the alpha-helical domain of the Galpha subunit in determining GDI sensitivity. I defined the structural features of the Gialpha2 alpha-helical domain that mediated the insensitivity of Gialpha2 to RGS14 GDI activity. This study revealed a previously unappreciated level of specificity in G protein regulation and further defined the likely physiological partners of RGS14.