Genetic identification of residues involved in association of alpha and beta G-protein subunits.

Abstract

The GPAI, STE4, and STE18 genes of Saccharomyces cerevisiae encode the a, jI, and y subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and AL138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpal protein. A mutation at position E307 of the Gpal protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant a subunit to physically associate with a specific mutant GoI subunit. The mutation in the Gpal protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the a subunit with the wild-type ,1 subunit. Yeast cells constructed to contain only the mutant a and , subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified Gat subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.Heterotrimeric G proteins function to transmit signals from a variety of cell surface receptor proteins to intracellular signaling components through a common molecular mechanism (32). The at and 13y subunits of heterotrimeric G proteins undergo an association-dissociation cycle in response to the interaction between the cell surface receptor and the receptor's cognate ligand. This association-dissociation cycle is regulated by guanine nucleotides. Unstimulated receptors are coupled to nonactivated G proteins in which the subunits are associated, and GDP is bound to the a subunit. The ligandbound receptor stimulates exchange of the bound GDP for GTP, and this exchange triggers subunit dissociation; subsequent GTP hydrolysis allows reassociation of the subunits and a return to the resting state. This cycle predicts that the at subunit of G proteins is capable of undergoing a conformational change which modifies its affinity for the 13y subunit (9).We have used a genetic approach to identify specific regions of the G protein a and 1 subunits that are important for their proper association with each other. The strategy of defining molecular interactions through genetics can supplement or even correct direct structural studies and can provide a high level of resolution (13,33,45). Previous approaches to investigating the associations among G-protein subunits have shown that deletion of the amino terminus of Gao blocks association of the Ga and GP subunits (10), while mutations in the switch box of Gs ax prevent a-1 dissociation in response to ligand stimulation of receptor (23). Furthermore, mutation of Cys-215 of Go a prevents cross-linking to 0-y, although ...

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

“…To ascertain the specificity of the peptide effect, we substituted the residue corresponding to Met-101 in G␤ to Asn. This Met is conserved in most G␤ from different species (12) and mutation of the residue at this position in yeast abolishes G␣ interactions (14). The ''mutated'' peptide (m-TTN) containing Asn at the position corresponding to G␤-101 was much less efficacious than the TTN peptide in inhibiting G␤␥ stimulation (Fig.…”

The WD repeats of Tup1 interact with the homeo domain protein alpha 2.

“…Second, antibodies raised to peptides within the repeats of [3-transducin can inhibit its ability to interact with the transducin ~ subunit (Murakami et al 1992). Third, Gpal and Ste4, the c~ and B subunits of a yeast G protein, interact in vivo in the two-hybrid fusion assay; this interaction is disrupted by mutations in the second WD repeat of Ste4 (Clark et al 1993;Whiteway et al 1994). In this study we have shown directly that a single WD repeat of Tup 1 will bind to a2 and can therefore function as a discrete unit.…”

The Mating-Specific Gα Protein of Saccharomyces cerevisiae Downregulates the Mating Signal by a Mechanism That Is Dependent on Pheromone and Independent of Gβγ Sequestration

“…First, the two-hybrid assay (5, 15) was used to quantitate the relative affinities of wild-type Gpa1, E364K, and G322E for G ␤ (Ste4). Clark et al (7) and Whiteway et al (49) have previously used this method to examine the interactions between Gpa1 and Ste4 and between Ste4 and Ste18. Their work demonstrated the utility of the two-hybrid assay in assessing G-protein subunit interaction.…”