binding (Bourne et a l 1991).
The recent revelation o f the crystal structure o f GTP- and GDP-liganded transducin and a n
(N oel et a l 1993, Lambright et al 1994, Coleman et a l 1994) has allow ed further insight into the structural basis o f heterotrimeric G protein function.
The heterotrimeric G^ and Gji a subunits consists o f two domains; one, a GTPase domain that contains the guanine nucleotide binding pocket as w ell as sites for binding receptors, effectors, and By. The GTPase domain is similar to that o f p21^^ and consists o f a six- stranded B sheet (B1-B6) surrounded by a set o f five helices ( a l- a 5 ) . A second highly a - helical domain unique to heterotrimeric G proteins is also present, but its function is less clear. The highly helical domain comprises seven helices, (helix A -helix G), and is linked to the GTPase domain by two extended linker strands (Figure 1.5). Betw een the tw o domains
lies a deep cleft w ithin w hich the guanine nucleotide is tightly bound. The helical dom ain, in particular the helix B-helix C region, may also contribute to the effector-binding site (C olem an et al 1994). There is a high level o f am ino acid sequence diversity betw een G a^ and G a il the helix B -helix C regions, such divergence m ay specify the differential effector interactions o f these tw o subtypes o f G protein. Substantial structural changes also occur in the helix B-helix C segm ent upon GTP hydrolysis (C olem an et al 1994).
[Tl
—\ / Helical Domain ^ n__cc-'-11-;
S .iic h ll GTPase Dom |[b. - ( ^ r
ÎS..
Helical Domain ^ / ^ GTPase DomainFigure 1.5 Three-dim ensional crystal structure of Ga^ in the GDP- (a) and G TP-bound (b) forms
(Lamhright et al 1994)
1.6,2 G u a n in e-n u cleo tid e E x c h a n g e Ga^, Ras and EF-Tu differ in their affinity to bind guanine nucleotides. Exchange factors accelerate nucleotide exchange in both R as and EF-Tu, but a significant basal exchange rate persists in their absence (review ed in B oguski & M cC orm ick 1993). Exchange does not occur for transducin in the absence o f activated rhodopsin. The structural feature that distinguishes G a^ from both Ras and E F -T u is the large helical dom ain that forms over one wall o f the nucleotide-binding cleft and thereby occludes the nucleotide (Noel et al 1993). A ctivated rhodopsin m ay open the nucleotide binding cleft to facilitate G D P/G TP exchange. The stereochem ical links betw een the receptor-binding surface and elem ents that clam p the nucleotide are structurally interdependent so that a receptor-triggered structural change may initiate a cooperative unravelling o f the interactions that secure the nucleotide. Follow ing release o f G D P, GTP binds to the open nucleotide-binding cleft. Crystal analysis studies reveal that the new ly bound Y phosphate initiates a conform ational change, w hich potentially alters the structure o f the region corresponding to am ino acids 198-215 (com prising the a 2 helix) (Figure 1.5), and leads to dissociation from the activated receptor and By heterodim er (Noel et al 1993). M utations in the corresponding region o f G ag also indicate that this region is critical in regulating affinity for Gfjy (Lee et al 1992).
i.6 .5 G TPase A ctivity The polypeptide has a low intrinsic GTPase activity
^ ca t ^ 0.03 min”l) but in the presence o f the GTPase activating protein (GAP) the GTPase activity is increased 200-fold (Trahey & McCormick 1987). In contrast, the a subunit o f heterotrimeric G-proteins have a relatively high intrinsic GTPase activity (k^^^2-3 m in“l) (Bourne et al 1990). The region in G protein a subunit polypeptides responsible for the high GTPase activity has putatively been mapped to a region that surrounds Arg-201. This site is located in the middle o f the ccg polypeptide and is the residue ADP-ribosylated by cholera toxin (Casey & Gilman 1988). ADP-ribosylation o f Arg-201 results in inhibition o f the intrinsic ocg GTPase activity. Additionally, mutation o f Arg-201 also results in inhibition o f the intrinsic GTPase activity, despite the fact that this region is not involved in GTP/GDP binding (Casey & Gilman 1988). It has been postulated that this region o f the polypeptide is controlling a GAP function which is intrinsic to G protein a subunit polypeptides (Landis et a l 1989). Residues surrounding Arg-201 share hom ology with a putative GAP-binding site in p21^^ (McCormick 1989). The intrinsic GTPase activity o f transducin and G q/n is stimulated directly by their respective effector proteins, cGMP phosphodiesterase and phospholipase-CB (Arshavsky et a l 1991, Bernstein et a l 1994). These effector proteins may also interact with a GAP-binding site on the a polypeptide o f the heterotrimeric G proteins, in a similar manner as the p21^^ GAP, to promote GTP hydrolysis.
The highly helical domain o f G a |i and transducin, contain a key residue (Arg-174 in a^, Arg-178 in a j i ) that may be needed for GTP hydrolysis and regulation o f GTPase activity (N oel et a l 1993, Coleman et al 1994). Arg-178, which is located on linker 2, and Gln-204, w hich is located on the
0.2 helix o f the a j j subunit polypeptide are critical for GTP hydrolysis (Colem an e t al
1994). Similarly, Arg-174 and Glu-203 on the polypeptide have also been implicated in GTP hydrolysis (N oel et a l 1993). Mutations o f the corresponding Arg residue in G aj and Gocg dramatically reduces their respective GTPase activities and constitutively activates their respective target effectors (Landis et al 1989, Gibbs et a l 1990). A lso, this arginine on Gocg is the target for ADP-ribosylation by cholera toxin, which blocks GTP hydrolysis (Van Dop et a l 1984).
The weak hydrolytic activity o f p21*'^ , which does not have an Arg residue at a position analogous to 178 suggests that Arg-178 provides the energy to stabilise the transition state for GTP hydrolysis. The higher GTPase activity rates in heterotrimeric G proteins a - subunits may reflect an inherently better-structured a2-helix, which provides a properly positioned general base without the 2iid o f a GTPase-activating protein, and the presence o f
a transition-state-stabilising interaction with the side chain o f a conserved arginine, corresponding to Arg-174 and Arg-178. Since a 2 is structurally coupled to effector binding sites, Glu 203 and Glu 204 could be productively oriented by bound effectors to facilitate GTP hydrolysis (Arshavsky & Bownds 1992, Bernstein et a l 1992).
1 ,6 .4 R ecep to r a n d E ffector Interaction Pertussis toxin catalyses the ADP-ribosylation o f a j but not oig (Gilman 1987). Within the a jj polypeptide Cys-351, four amino acids from the C-terminus, is the residue ADP-ribosylated by pertussis toxin. Efficient pertussis toxin- catalysed a j ADP-ribosylation also requires the presence o f the By subunit com plex and sequences within the N-terminal moiety o f the a subunit polypeptide (W oon et a l 1988). Chimeric studies and specific point mutations have revealed the C-terminal m oiety o f heterotrimeric G protein a subunits to encode the effector activation and receptor selectivity domains (Russell & Johnson 1993). The G4 and G5 sequences within the GDP/GTP binding domains are also encoded within the C-terminal moiety. Consistent with the receptor selectivity site mapping to the extreme C-terminus, the - cysteine^residues from the C-terminal end o f aj-like polypeptides is the amino acid ADP-ribosylated by pertussis toxin. The ADP-ribosylation o f this residue functionally uncouples receptor activation o f the covalently modified G protein a subunit.
The extreme C-terminal region, the aS helix and the N-terminal region are important sites o f interaction with receptors (Conklin & Bourne 1993, Neer 1994). Mutations in the C- terminus (Hirsch et al 1991), its covalent modification by PTX (W est et a l 1985), and peptide-specific antibodies raised against it (Gutowski et a l 1991) all uncouple G-proteins from their associated receptors. The region encoded by residues 311-328, which are located on the B strand (B6) linking the 4th and 5th a-h elices o f (Lambright et a l 1994) is also
involved in receptor interaction. Like the C-terminal decapeptide, a peptide raised against this region blocks activation o f Gt by its receptor photorhodopsin (Hamm 1991). An N - terminal peptide also inhibited interaction o f Gt with photorhodopsin (Hamm et a l 1988). A n activated receptor triggers the intracellular response by decreasing the affinity o f the a subunit for GDP, perhaps by promoting a conformational change in the C-terminal a helix. This effect is mimicked by deletion o f 14 amino acid residues from the C-terminus o f olq
(Denker et a l 1992). A conformational change occurring in the C-terminus o f the a 5 helix may be transmitted to the N-terminal loop. Mutations o f amino acid residues in the N - terminal loop also decrease GDP affinity (Thomas e t a l 1993, liri et a l 1994).
It w ould appear that the C-terminus o f the a subunit polypeptide confers the specificity o f receptor interaction. U se o f a subunit chimeras has enabled mapping o f certain a regions as receptor interaction sites. For example, when the a portion o f the C-terminus o f Gocq is replaced by the corresponding region o f Gi2, then Gq is able to couple with receptors normally associated with G{2 (Conklin et a l 1993). However, since many G protein a subunits are identical at the C-terminus but couple to different receptors, other regions o f the a polypeptide may have a role in regulating receptor interaction specificity. Lee et al
(1995) have shown that the region in the middle o f the G a j^ m olecule (corresponding to residues 220-240), and some elements in the amino-terminus, in combination with the C-
terminus is critical in maintaining interaction specificity with the chemoattractant factor receptor.
The effector-binding region has been mapped only for the pairs ocg/adenylate cyclase and a j/cGMP phosphodiesterase (reviewed in Conklin & Bourne 1993).M utagenesis o f ocg revealed three regions involved in coupling Gs to its effector adenylate cyclase (Berlot & Bourne 1992). The key effector regions were found to be located on the loops a2-B 4, a3-B5, a4-B6. O f these, only the a2-B4 loop exhibits a clearly identifiable structural change on nucleotide exchange (Lambright et al 1994) (Figure 1.5). The effector binding region o f ocg also partially overlaps the putative By-binding surface (Lambright et a l 1994). It is therefore unlikely that the a subunit can simultaneously bind effector and By.
1.6 ,4 fiy S u b u n itln te ra c tio n The By subunits, effectors, and receptors appear to bind to different surfaces o f a subunits. The first 25 amino acids o f the a subunit are essential for By binding (Fung & Nash 1983, Denker et a l 1992), but their position in 3-D structure is unknown since they are mobile and therefore do not show in the crystal (Coleman et a l 1994). The By-binding surface probably also includes the a 2 helix o f the GTPase domain since a cysteine residue on this helix (C ys-215 in ocq) can be chemically crosslinked to By (Thomas et al 1993). Since binding o f By depends critically on the nucleotide bound to the a subunit, it makes sense that the By contact surface w ould include a region such as the a 2 helix that is different in the GDP- and GTP-liganded states (Lambright et a l 1994). The N-terminal m oiety o f ccg has been shown to control both GDP dissociation and interaction with the By subunit (Conklin & Bourne 1993). Similarly, a monoclonal antibody directed against the N-terminus o f G^ causes G^xt to dissociate from By (M azzoni e t a l 1989). N-terminal myristoylation o f Œq enhances its affinity for binding By (Linder e t a l 1991 ), and an N-terminally myristoylated peptide com petitively inhibits binding o f to By (Kokame et al 1992).