Browsing by Subject "GTP-Binding Protein alpha Subunits"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item Allosteric Determinants of Guanine Nucleotide Binding Proteins and Methods to Crystallize the Cytosolic Domains of Adenylyl Cyclase(2004-05-04) Hatley, Mark Edward; Gilman, Alfred G.The cytosolic domains of mammalian adenylyl cyclases, termed C1 and C2, are responsible for catalytic activity and most regulatory properties. Crystal structures of the soluble catalytic core of adenylyl cyclase bound to activators Gsa and forskolin were previously determined. However, structural information regarding low activity (non-Gsa or forskolin bound) states of the enzyme is lacking. Genetic and biochemical methods were utilized to overcome the low affinity of the cytosolic domains in the absence of activators. A genetic screen in Saccharomyces cerevisiae identified mutations that activate mammalian adenylyl cyclase in the absence of Gsa. The increased affinity of the K1014N-C2 mutant protein for the C1 domain in the absence of Gsa was exploited to isolate a complex containing C1 and C2 in the absence of Gsa. Unfortunately, this complex crystallized but failed to diffract due to heterogeneity. Intein-mediated protein ligation and expression of a C1-C2 fusion protein in adenylyl cyclase deficient Escherichia coli were explored to circumvent the low affinity of the domains. However, the yields of products were insufficient for crystallization. Members of the G protein superfamily contain nucleotide-dependent switches that dictate the specificity of their interactions with binding partners. Using a new sequence-based method termed statistical coupling analysis (SCA), I identified the allosteric core of these proteins - the network of amino acid residues that couples the domains responsible for nucleotide binding and protein-protein interactions. One-third of the 38 residues identified by SCA were mutated in the G protein Gsa, and the interactions of GTPgamma S- and GDP-bound mutant proteins were tested with both adenylyl cyclase (preferential binding to GTP-Gsa) and the G protein beta gamma subunit complex (preferential binding to GDP-Gsa). A two-state allosteric model predicts that mutation of residues that control the equilibrium between GDP- and GTP-bound conformations of the protein will cause the ratio of affinities of these species for adenylyl cyclase and beta gamma to vary in a reciprocal fashion. Observed results were consistent with this prediction. The network of residues identified by the SCA appears to comprise a core allosteric mechanism conferring nucleotide-dependent switching; the specific features of different G protein family members are built upon this core.Item Multiple Gq-Coupled Receptors Converge on a Common Protein Synthesis-Dependent Long Term Depression That Is Affected in Fragile X Syndrome(2007-12-18) Volk, Lenora Joanne; Huber, Kimberly M.Activation of Gq-coupled group I metabotropic glutamate receptors (mGluR1, mGluR5) induces long-term synaptic depression (LTD) that requires rapid, dendritic protein synthesis. The significance of protein synthesis-dependent mGluR LTD to cognitive function is highlighted by the recent finding that mGluR-dependent LTD is enhanced and protein synthesis-independent in the mouse model of fragile X syndrome mental retardation (FXS, Fmr1 KO mice). In fact, group I mGluR antagonism ameliorates some symptoms of FXS in model organisms. However, disagreement exists in the literature as to the specific roles of mGluR1 and mGluR5 in LTD. Using pharmacological and genetic manipulations, I find that mGluR1 or mGluR5 activation is sufficient to induce LTD. In contrast, I see a selective role for persistent mGluR1 activity in expression of LTD induced with the group I mGluR agonist, DHPG. These data demonstrate a novel role for mGluR1 in induction and expression of LTD at hippocampal Schaffer collateral-CA1 synapses and confirm a role for mGluR5 in induction of LTD at this synapse. LTD induced synaptically with paired-pulse low frequency stimulation (PP-LFS) is Gq- and protein synthesis-dependent and shares common signaling and expression mechanisms with DHPG-induced LTD. However, I find that PP-LFS LTD persists in the presence of group 1 mGluR antagonists and in mGluR1 or mGluR5 knockout mice. These data led to the hypothesis that Gq-coupled receptors other than mGluRs are activated by PP-LFS to induce LTD, and LTD mediated by these receptors should share similar signaling and expression mechanisms with mGluR LTD. A previous study shows that muscarinic acetylcholine receptors (mAChRs) activate protein synthesis in hippocampal CA1 dendrites. Data presented here demonstrate that PP-LFS activates both Gq-coupled mAChRs and mGluRs to induce LTD. Pharmacological activation of mAChRs induces LTD that requires rapid protein synthesis and activation of ERK and mTOR translational activation pathways. New proteins maintain mAChR-dependent LTD through a persistent decrease in surface AMPA receptors. In addition, mAChR LTD is enhanced and protein synthesis-independent in Fmr1 knock-out mice. These data reveal that multiple Gq-coupled receptors converge on a common protein synthesis-dependent LTD mechanism that is aberrant in FXS. These findings suggest novel therapeutic strategies for FXS in the form of mAChR antagonists.Item Regulation of Guanine Nucelotide Exchange in Inhibitory G Protein Alpha Subunit by Activator of G Protein Signaling 3 and Novel Regulatory Peptides(2005-12-19) Adhikari, Anirban; Sprang, Stephen R.The release of GDP from the nucleotide binding pocket of G protein a subunit (Ga) is accelerated by guanine nucleotide exchange factors (GEFs) and inhibited by guanine nucleotide dissociation inhibitors (GDIs). The beta gamma subunit of heterotrimeric G protein and GoLoco motif are GDIs. Activator of G protein signaling 3 (AGS3), a GDI containing four GoLoco motifs in its C-terminal domain (AGS3-C), binds specifically to GDP-bound Gai1 and inhibits the binding of GTP to Gai1. The stoichiometry and dissociation constant for the binding of AGS3-C to Gai1, determined using isothermal titration calorimetry (ITC), indicates the presence of two apparent high affinity (Kd ~ 20 nM) and two apparent low affinity (Kd ~300 nM) binding sites for Gai1. Upon deletion of the C-terminal GoLoco motif from AGS3-C, the remaining sites are approximately equivalent with respect to their affinity (Kd ~ 400 nM) for Gai1. Peptides corresponding to each of the four GoLoco motifs of AGS3 (referred to as GPR1-4, going from the N to C terminus) bind to Gai1 with Kd values in the range of 1-8 muM. GPR1, GPR2, and GPR4 were active as GDIs, but GPR3 was inactive. However, addition of N and C terminal flanking residues to the GPR3 GoLoco core increases its affinity for Gai1 and conferred GDI activity to the level of AGS3 itself. R6A is a Gai1 GDP selective peptide that has GDI activity towards Gai1 despite no sequence similarity with the GoLoco motif. The 1.8 ?ystal structure of Gai1 GDP:R6A complex shows that switch II region of Gai1, which is disordered in the GDP bound state, adopts an ordered structure through its direct interactions with R6A. Binding of R6A orients residues 202 to 204 of switch II (GG-loop) towards the nucleotide binding pocket and within 3 ? switch I, which suggests that R6A mediates its activity by occluding the potential exit route of GDP. The structure of Gai1 GDP:R6A, when compared to the structures of GoLoco and G beta gamma bound Gai1 GDP, reveals similarity in the conformation of GG-loop among the GDI bound structures of Gai1. A mutation in R6A, designed to position the GG-loop away from the nucleotide binding pocket in Gai1 GDP:R6A complex, abolishes the GDI activity and instead confers GEF like activity to R6A. Thus, the conformation of the GG-loop is likely to be a crucial structural determinant of the rate of GDP release from Gai1.