Structural characteristics of Ga subunits

Ga subunits share sequence homology with p21-Ras, transcription factor EF-Tu and other GTPases in their guanine nucleotide-binding pockets (Bourne etal. 1990, 1991). The initial model of Ga is a Ras-like structure with a relatively large insertion between the first two guanine nucleotide-binding regions (Conklin and Bourne 1993). Resolution of crystal structures of Gat1 and Gai1 confirmed the presence of the two structural domains as the helical and GTPase domains (Sprang 1997). The helical domain encompasses seven a helices (aA to aG; Figs 3.1 and 3.2a), with the first six forming the core of the helical domain. aG is actually located on the GTPase side facing the helical domain. Similar to monomeric G proteins, the GTPase domain is comprised of five a helices (including both terminal helices) and six P strands (Figs 3.1 and 3.2a). The conformations of both GTPase and helical domains are highly similar between the GDP- and GTP-bound Ga structures. A slightly more compact structure is observed in the GTP-bound form, with the two domains getting closer to each other to bury the GTP in the cleft. Residues lying on the interface between the two portions

Fig. 4.1 The GTPase cycle. Ligand (L) binds to a cognate G protein-coupled receptor (GPCR)-G protein complex and triggers the release of GDP from Ga. The empty state of Ga has low affinity to ligand-bound GPCR and permits the binding of GTP. The ligand-bound GPCR can elicit another round of G protein activation by promoting nucleotide exchange of more G protein trimers. GTP-bound Ga subsequently dissociates with G^y and both compartments stimulate various effectors. Intrinsic GTP hydrolysis, which can be accelerated by the binding of a regulator of G protein signaling (RGS), eventually turns off the Ga and release a phosphate (Pi). Finally, the GDP-bound Ga combines with G^y and ligand-free GPCR thereby returning to the resting stage.

Fig. 4.1 The GTPase cycle. Ligand (L) binds to a cognate G protein-coupled receptor (GPCR)-G protein complex and triggers the release of GDP from Ga. The empty state of Ga has low affinity to ligand-bound GPCR and permits the binding of GTP. The ligand-bound GPCR can elicit another round of G protein activation by promoting nucleotide exchange of more G protein trimers. GTP-bound Ga subsequently dissociates with G^y and both compartments stimulate various effectors. Intrinsic GTP hydrolysis, which can be accelerated by the binding of a regulator of G protein signaling (RGS), eventually turns off the Ga and release a phosphate (Pi). Finally, the GDP-bound Ga combines with G^y and ligand-free GPCR thereby returning to the resting stage.

of Ga form specific intramolecular interactions that are crucial for controlling the nucleotide exchange rate, basal activity, and receptor-triggered activation of G protein.

The guanine nucleotide-binding pocket of Ga subunit is comprised of five stretches of well-conserved amino acids (Bourne et al. 1991; Fig. 4.2), spreading along the Ga sequence but clustering in a deep cleft between the GTPase and helical domains. Mutations located in the nucleotide-binding regions produce mutants that serve as useful tools for investigating the structure-function relationships of G proteins. For example, mutations of the glutamine residue in the third nucleotide-binding region signature DVGGQR of virtually all Ga subunits produce constitutively active phenotypes, and spontaneous occurrence of these mutations are associated with a number of endocrine tumours (Lyons et al. 1990). Such mutations have been successfully employed to define the specific functions of various Ga subunits in regulating effectors and cell growth. The two glycines preceding the

GTPase domain

Helical domain

GTPase domain

Helical domain a2 a3 aG a4 a5

a2 a3 aG a4 a5

Secondary structures

Switch regions GPy binding sites GDP/GTP binding sites Receptor interaction Effector interaction

Fig. 4.2 Structure and functional domains of Ga. Various secondary and tertiary structural elements of Ga are schematically described. The nomenclature is according to the first crystal structure of GTPyS-Gat1 and depicted with different shapes and shadings. L1 and L2 are the two linker regions between helical and GTPase domains. Switch regions and other functional regions interacting with various molecular partners are indicated at the bottom. Most of them are discrete regions spanning along the primary structure of Ga subunit, but they are close together in the three-dimensional structures (see Fig. 3.2). Effector interacting regions are relatively diverse among various Ga subunits, but mainly within the C-terminal 40% of the primary structure.

glutamine ensure flexibility of the third nucleotide-binding region for accommodating the GTP-induced conformational changes. Mutation of either one of the glycine residues in Ga creates a dominantly negative phenotype (Osawa and Johnson 1991).

Upon activation, several regions of the Ga undergo dramatic conformational changes. The first three switches, denoted as Switch I, II, and III, cluster in the GTPase domain and a discrete Switch IV is observed in the helical domain of Ga¡i but not Gati (see Sprang 1997; Fig. 4.2). These switch regions are involved in a series of concerted movements that facilitate nucleotide exchange and GTP hydrolysis. The first three switches overlap with two important nucleotide-binding regions, as well as a major binding surface for G^y and effector molecules (Fig. 4.2). The binding of GTP lifts the Switch I upward and the strictly conserved threonine (Thr-177 of Gat1) residue is oriented to the proximity of the y-phosphate of GTP. Switch II covers the ^3/a2/^4 structures. Extensive electrostatic and hydrophobic interactions are found between Switches II and III in the GDP-bound state. These are disrupted and several ionic bridges are re-established during activation for stabilizing the GTP-induced conformation. As compared to GTPyS-bound Gat1, the aB/aC loop of GTPyS-bound Ga¡1 is packed more tightly. This region is denoted as Switch IV because significant conformational changes occur during GTP hydrolysis. Xenopus expresses a Gas mutant with six amino acids different from the human homologue. The mutation maps to a region corresponding to the Switch IV of Ga¡1 and this mutant exhibits a reduced guanine nucleotide exchange rate and does not activate AC (Echeverría etal. 2000).

The N-terminus of Ga is a single helical structure denoted as aN that stretches out from the main body and embraces the G^y complex (Fig. 4.3). Both the N-terminus of Ga and the C-terminus of Gy are adjacent to each other. Although there is no observable contact

GTPase domain

GTPase domain

Helical domain

Helical domain

Fig. 4.3 Ribbon structural models of trimeric G protein and its receptor. The three-dimensional arrangements of the structural elements of (a) GDP-Mg2+-bound Gat1 subunit, (b) Gp1y1 complex, and (c) rhodopsin and trimeric Gt1 are illustrated. In (a), the N-terminal helix is omitted (but appears in (c)). The bound GDP (CPK scheme) and Mg2+ are displayed in space-filled models, which situates between helical and GTPase domains. Switch regions are marked with inverted letters in circles. In (b), the termini of both Gp (Np and Cp) and Gy (NY and CY) are indicated and the seven blades are numbered as in Neer and Clapham (1997). Most of the receptor- and effector-interacting residues of Gp subunits are on blades 1-3, as indicated. In (c), the structural models of 11-c/s-retinal-bound rhodopsin (Palczewski etal. 2000) and trimeric Gt1 (Ga, Gp and gy; Lambright et al. 1996) are assembled together according to the proposed receptor-interacting surface as described (Lichtarge et al. 1996; Bourne 1997; Hamm 1998). Switch regions I—IV of Ga subunit are marked similarly as in (a). Coordinates of all the structural models are obtained from the Protein Data Bank (Berman et al. 2000; http://www.rcsb.org/pdb) and visualized by Swiss PDB Viewer Deep View Version (Guex and Peitsch 1997; http://www.expasy.org/spdbv/) and POV-Ray 3.1 for rendering (http://www.povray.org).

between Ga and Gy subunits, the close proximity of their two terminal regions supports the idea that the lipid moieties attached on them may strengthen the association of the trimer. The N-terminus of Ga is generally accepted as an essential GPy-binding region and the large loop structure extending from blade 2 of GP helps to orientate the aN helix along the side of GP (Fig. 4.3). Another major GPy-interacting surface has been mapped to Switches I and II of the Ga subunit. Within Switch II of Ga in the trimer crystals, the a2 helix adopts a loose 3io helical structure resembling that observed in the GDP-bound monomer. Residues of the a2 helix swing outward and interact with GP. In the GTP-bound state, conformational changes in Switch I and Switch II regions of Ga disrupt most of the intermolecular interactions with GPy resulting in the release of the GPy complex.

Lipidation of Ga subunits occur at the N-terminus. Myristate is co-translationally incorporated at Gly2 of the Ga;-subfamily members and is relatively stable (Table 4.1). The myristoylated Ga subunits have the N-terminal methionine deleted during the modification. Gat1 can be exceptionally modified with other fatty acids such as stearate, laureate, and oleate which are believed to occur specifically in photoreceptor cells (Johnson et al. 1994). N-myristoylation facilitates the membrane anchorage of Ga, increasing its affinity for GPy and enhancing the regulation of AC (Wedegaertner et al. 1995). Reversible palmitoylation occurs on the cysteine residues at the N-terminus of almost all known Ga subunits through a thioester linkage to the side chain sulfhydryl group (Table 4.1). The same cysteine residue can be arachidonylated but its functional impact is unclear. Strikingly, Gaj1, Gao, and Gaz can undergo autoacylation in the presence of palmitoyl-coenzyme A (or other fatty acyl derivatives) at physiological pH and temperature (Duncan and Gilman 1996). The rate of autoacylation can be enhanced by the addition of GPy complex or increasing pH. The reversibility of palmitoylation is considered as a regulatory handle on the Ga, and a palmitoylation/depalmitoylation cycle of Gas has been proposed (Wedegaertner and Bourne 1994). Upon receptor activation, nucleotide exchange in Gas promotes its dissociation from GPy. The GTP-bound Gas is rapidly depalmitoylated by a cytosolic palmitoylesterase and then translocates to cytosol. After GTP hydrolysis, Gas is recruited by GPy and returns to the plasma membrane, where the palmitoyltransferase replenishes Gas with palmitate moiety to form fully acylated Gs protein.

Many Ga subunits are substrates for kinases (Table 4.1) and phosphorylation exerts different functional impacts on various Ga subunits. Gat1 is the first Ga subunit found to be phosphorylated by both protein kinase C and insulin receptor kinase. Src kinase can phosphorylate Gas at Tyr37 and Tyr377. For Gaq, phosphorylation of Tyr356 is essential in type 1a metabotropic glutamate receptor-mediated activation. Epidermal growth factor receptor (EGFR) is a membrane-bound tyrosine kinase that appears to phosphorylate and activate both Gas and Gai1-2. Protein kinase C phosphorylates Gai2, Gaz, Ga12, and Ga13, and Ga16. Uniquely, Gaz can be phosphorylated by p21-activating kinase 1. Associations of Gaz and Ga12 with GPy subunits and other molecular partners are impaired following phosphorylation of their N-terminal helices, whereas phosphorylation of Ga16 diminishes thyrotropin-releasing hormone receptor-induced Cl- current in Xenopus oocytes (reviewed in Chen and Manning 2001).

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