Ga Subunits

Ga subunits are members of the Ras superfamily, which also includes translation elongation factors and the components of the signal recognition apparatus. In mammals, the family of Ga isoforms is encoded by 16 genes; these can be sorted into four closely related homology groups or classes named for representative members of each class: Gas, Ga;, Gaq, and Ga12 (Fig. 1). Two variants of Gas are generated by alternative mRNA splicing. Each member of the Ga family interacts specifically with one effector or effector isoform, although certain effectors are regulated by more than one species of Ga. Known effectors include all isoforms of AC: Gas, Gaolf, and Ga; (a negative regulator of types I and V AC) [5]; PDE: Gat; PLCP isoforms: Gaq class members [6]; and p115RhoGEF: Ga13 [7]. Effectors of certain Ga proteins, Gao, and Gaz, remain in question.

Ras superfamily proteins are built upon a scaffold of six parallel P-strands, layered on each side by a set of five a-helices (Fig. 2). Unique to the heterotrimeric Ga family is an a-helical bundle domain inserted into the loop between

Table I Selected Structures of Heterotrimeric G Proteins and Their Complexes

Protein

Nucleotide

Ref. and PDB code

Gat

Mg2+»GTPySa

[15] 1TND

Gai1

Mg2+»GTPyS

[13] 1GIA

Gai1

Mg2+»GppNHpb

[65] 1CIP

Gas

Mg2+»GTPyS

[17] 1AZT

Gaii

Mg2+*GDP*AlF4-

[13] 1GFI

Gat

Ca2+»GDP»AlF;-

[25] 1TAD

Gai1 (G203A)

GDP»Pi

[66] 1GIT

Gaii

Mg2+»GDP»SO4-

[67] 1BOF

Gat

Mg2+»GDP

[16] 1TAG

Gai1

GDP

[14] 1GDD

Gai1»Gßi»Gy2

GDP

[45,49] 1GG2

Gat»Gßi»Gy1c

GDP

[48] 1GOT

Gat»GoLocod

GDP

[68] 1KJY

Gßi'Gyi

[47] 1TBG

Gßi»Gy1 »Phosducin

[69,70] 1AOR,1B9X

Gai1»RGS4

Mg2+»GDP»AlF4-

[35] 1AGR

Gat»RGS9

Mg2+»GDP»AlF4-

[20] 1FQK

Gat»RGS9»PDEye

Mg2+»GDP»AlF4-

[20] 1FQJ

Gas»ACf

Mg2+»GTPyS

[19] 1AZS

Gas»ACg

Mg2+»GTPyS

[71] 1CJU

aGTPyS-guanosine 5'-[y-thio]triphosphate. bGppNHp-guanosine-5'-(Py-methylene)triphosphate.

cGa subunit is a chimera comprising residues 26 to 215 of bovine Gat, residues 220 to 298 of rat Ga^ and residues 295 to 350 of bovine Gat.

GoLoco motif peptide from RGS14.

ePDEY-cyclic GMP phosphodiesterase Y subunit. f

AC: a complex between the C1 domain of adenylyl cyclase type V and the C2 domain of adenylyl cyclase type II. These domains comprise the catalytic unit. A soluble forskolin derivative is bound at the regulatory site of AC. The domains adopt the open conformation.

gThis complex contains the ATP analog P-L, 2',5', dideoxy adenosine triphosphate, and two magnesium ions. The domains adopt a closed conformation.

aGTPyS-guanosine 5'-[y-thio]triphosphate. bGppNHp-guanosine-5'-(Py-methylene)triphosphate.

cGa subunit is a chimera comprising residues 26 to 215 of bovine Gat, residues 220 to 298 of rat Ga^ and residues 295 to 350 of bovine Gat.

GoLoco motif peptide from RGS14.

ePDEY-cyclic GMP phosphodiesterase Y subunit. f

AC: a complex between the C1 domain of adenylyl cyclase type V and the C2 domain of adenylyl cyclase type II. These domains comprise the catalytic unit. A soluble forskolin derivative is bound at the regulatory site of AC. The domains adopt the open conformation.

gThis complex contains the ATP analog P-L, 2',5', dideoxy adenosine triphosphate, and two magnesium ions. The domains adopt a closed conformation.

the first and second P strands of the Ras-like domain. Ga subunits are modified by N-terminal myristoylation [8] (Gat) and thioester-linked palmitoylation (Gas, Gaq, Ga13), or both (Ga;, Gao, Gaz) [9,10]. The latter confers plasma membrane localization upon Gas and Gaq but may be reversed upon activation [11]. Myristoylation is required in some cases for activity, for example, efficient inhibition of adenylyl cyclase by Ga;1 [12].

GTP is bound between the helical and Ras-like domains but interacts primarily with conserved sequence motifs within the Ras domain [13-17]. The P-loop, which enfolds the alpha and beta phosphates of the nucleotide, contains a characteristic Walker A sequence motif, Ga/tGESGKST [18], which is permissive for the tight turn required to encompass the phosphate. The lysine residue is a critical P-phosphate ligand and the following serine residue binds the catalytic Mg2+ ligand. The connector leading from the helical domain to the Ras-like domain contains a series of residues called Switch I. The arginine residue (178 in Ga;1) within this sequence (...RVXTTG...) is an important catalytic ligand, and the succeeding threonine is the second Mg2+ ligand (Fig. 3). The gamma phosphate group of GTP is cradled by a tight turn (...DVGGQ...) which precedes Switch II, an irregular and conformationally mobile helix (a2). The glut-amine residue in this series (204 in Ga;1) plays a critical catalytic role in GTP hydrolysis. However, in the structures of Ga subunits bound to slowly hydrolyzable GTP analogs, the catalytic glutamine and, in Gai1, the catalytic arginine as well are either poorly ordered or adopt conformations in which they would be incapable of providing catalytic assistance (Fig. 3). A key role of GTP, in league with Mg2+, is to maintain the conformational state and structural integrity of the helical Switch II via a set of hydrogen bonds and oxygen-metal interactions that link Mg2+^GTP with the P-loop, Switch I, and Switch II. Structural studies of Ga;1 and Gat show that, in the guanosine diphosphate (GDP) state, Switch II is either wholly disordered or adopts an alternate conformation. The well-ordered state induced by GTP also

Figure 1 A phylogenetic tree, using CLUSTAL_W(81) of the mammalian family of human Ga subunits.
Figure 2 Schematic of the GTP-bound complex of Gai1 with secondary structure elements (arrows: p-strands, coils: a-helices). GTP is shown as a ball-and-stick model. (From Sprang, S. R., Annu. Rev. Biochem., 66, 639-678, 1997. With permission.)

promotes a set of ionic contacts between Switch II and the (34-a3 loop called Switch III. Upon GTP hydrolysis, the network of interactions between the three switch regions is altered or lost.

The purine ring of the guanine nucleotide is cradled by two conserved loops, (35-aG and (36-a5 (Fig. 2). The aspartate residue within the first sequence (... FLNKKD...) confers specificity towards guanine nucleotides. The second loop acts in a supporting role. A variety of a mutations have been described, some of physiological relevance, that directly affect GTP hydrolysis, nucleotide specificity exchange, and effector coupling. Such mutants are useful for probing or controlling the action of G proteins in cell culture or in vivo (Table 2).

Ga-Effector Interactions

In the two Ga-effector complexes for which structures are known, Ga binds the effector in the same manner, even though the structures of the effectors themselves are quite different. The interactions are dependent on the "activated" state of Ga that is stabilized by GTP. In the complex

Figure 3 Close-up of the catalytic site of the Gai1^Mg2+^GppNHp complex, showing the side chains involved in catalysis and Mg2+ binding. Residues of Switch I are blue, the P-loop is green, and Switch II is pale yellow. (Adapted from Coleman, D. E. and Sprang, S. R., J. Biol. Chem., 274, 16669-16672, 1999.)

Figure 3 Close-up of the catalytic site of the Gai1^Mg2+^GppNHp complex, showing the side chains involved in catalysis and Mg2+ binding. Residues of Switch I are blue, the P-loop is green, and Switch II is pale yellow. (Adapted from Coleman, D. E. and Sprang, S. R., J. Biol. Chem., 274, 16669-16672, 1999.)

between Ga^GTPyS and the catalytic domains of adenylyl cyclase [19] and that between Ga t^GTPyS and the y subunit of PDE [20], the effector is bound at the cleft between Switch II and the a3-p5 loop of Ga (Fig. 4). The effector specificity of Ga is conferred both by side chains in the effector binding segments and the conformation of the polypeptide chain within them. For example, Gai1 inhibits the Gas-stimulated activity of adenylyl cyclase isoforms I and V, but does not bind to the Gaa activation site [21], possibly interacting at a dyad-related site in the C1 domain instead. The ability of Gai1 to discriminate its own from the Gas binding site is unlikely to be entirely due to the amino acid sequence of the Switch II and a3-^5 loops, because all but two amino acids in the adenylyl cyclase contact region are conserved between the two Ga subunits. The failure of Gai1 to act as an activator (or Gas as an inhibitor) may stem from differences between the two proteins in the spacing and orientation of the a3-^5 loop and the a4-^6 loop that buttresses it [17]. Indeed, the a4-^6 loop had been proposed, on the basis of mutagenesis experiments, to direct the specificity of Gai2 and Gas toward their respective binding sites on AC [22], even though the structure of the complex revealed no direct contact with effector. In its interaction with PDEy, Gat uses the same structural elements that Gas employs in contacting AC. Although the chemical basis of certain of the Ga-effector interactions are conserved, the amino acid sequence differences between Gat and other Ga subunits are sufficient to ensure specificity. There is structural and biochemical evidence to suggest that Gas stimulates adenylyl cyclase by controlling the relative orientation of its catalytic domains. Gat (transducin), on the other hand, sequesters an inhibitory subunit of cyclic GMP phosphodiesterase.

Table II Selected Mutations in Ga Subunits

Mutation

Ga

Structural element

Effect

Structure

Ref.

G49A

Gas

P-loop

Reduced GTPase rate

[72]

G42V

Gai1

P-loop

Reduced GTPase rate

Gai1, 1ASO, 1AS2, 1AS3

[73]

S54N

Gas

P-loop; Mg2+ ligand

Weak Mg2+ binding; reduced receptor activation; negative dominant

[74]

R201X

Gaii, Gas

Switch I Catalytic R

Reduced GTPase rate; activating mutant

[75,76]

G202T

Gao

Switch II

Dominant negative

G226L

Gas

Switch II Catalytic Q

Reduced rate of GPy release (prevents activation), loss of Mg2+ affinity

Gai1(G203A), 1GIT,

Gai1(G203A)ß1Y2 1GG2

[77,78]

Q227L

Gas, Gaii

Switch II

Abolishes GTPase activity

Gai1(Q204L), 1GIL

[72,79]

D273N

Gao

Switch II NKXD motif

Switches nucleotide specificity to XTP (in background of Q205L)

[80]

A366S

Gas Gaii

P6-a5 loop

Increases GDP release rate, decreases thermostability

Gai1(A326S), 1BH2

[63,64]

T325A V328A F332A

Gat

a5 (C-terminal helix)

Increases GDP release

[62]

fluoroaluminate (Mg2+^AlF4-1 ) [13]. Fluroaluminate (AlF3) and its hydrates mimic the y phosphate of GTP [23] and in the presence of GDP promote the activated state of Ga [24]. Structural studies of Ga;i and the Ga^GDP^Mg^AlF4-1 complexes demonstrate that AlF4-1 forms a hexacoordinate complex with a P phosphate oxygen of GDP and a water molecule (the presumptive nucleophile) as axial ligands, thereby approximating the pentacoordinate transition state for phos-phorolysis [13,25]. The structures show that, relative to the ground state (Fig. 2) the Switch I arginine and Switch II glut-amine must be substantially reoriented in order to stabilize the transition state. It is therefore possible that a conformational rearrangement within the active site corresponds to the kinetic barrier to GTP hydrolysis.

In some cellular contexts (for example, regulation of adenylyl cyclase by Gas), the rate of signal termination may indeed correspond to the intrinsic GTPase rate of the regulatory Ga. However, it is clear that other physiological responses decay much more rapidly following agonist withdrawal (for example, visual recovery after a light flash or deactivation of G protein-regulated K channels.) Rapid signal termination is achieved by RGS (regulator of G protein signaling) proteins, a family of proteins that have in common a homologous stretch of «120 amino acids termed the RGS-box [26]. RGS domains function as GTPase-activating proteins (GAPs) for Ga subunits [27,28]. Rate enhancement conferred by RGS ranges from 5- to 10-fold (for RGS9 regulation of Ga [29]) to well over 50-fold (for RGS4 stimulation ofGa;1 [30]). RGS proteins show varying degrees of specificity towards their Ga substrates [26], and most

Adenylyl Cyclase Mg2

Figure 4 Schematic of Gas»Mg2+»GTPYS bound to the catalytic domains of adenylyl cyclase. The Ras-like domain of Gas is rendered in charcoal and Switch II is red. The C1 and C2 domains of type V and type II AC, respectively, are tan and magenta. (Adapted from Tesmer, J. J. G. et al., Science, 278, 1907-1916, 1997.)

Figure 4 Schematic of Gas»Mg2+»GTPYS bound to the catalytic domains of adenylyl cyclase. The Ras-like domain of Gas is rendered in charcoal and Switch II is red. The C1 and C2 domains of type V and type II AC, respectively, are tan and magenta. (Adapted from Tesmer, J. J. G. et al., Science, 278, 1907-1916, 1997.)

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