Abstract
The identification of multiple G protein beta and gamma subunit subtypes suggests a potential diversity of beta gamma heterodimers, which may contribute to the specificity of signal transduction between receptors and effectors. The assembly of beta and gamma subtypes is selective. For example, gamma 1 can assemble with beta 1 but not with beta 2, whereas gamma 2 assembles with both beta isoforms. To identify the structural features of the beta and gamma subunits governing selectivity in heterodimer assembly, a series of nonisoprenylated chimeras of gamma 1 and gamma 2 was constructed, and their interaction with beta 1 and beta 2 was assessed by their ability to direct beta expression to the cytosol in cotransfected COS cells. All of the gamma 1/gamma 2 chimeras were capable of interacting with beta 1 as judged by the cotransfection assay. Chimeras containing gamma 2 sequence near the middle of the molecule between two conserved sequence motifs were capable of interacting as well with beta 2. Among 12 divergent residues in this region, it was found that replacement of three consecutive amino acids in gamma 1, Glu-Glu-Phe (residues 38-40), with the three corresponding amino acids of gamma 2, Ala-Asp-Leu (residues 35-37), conferred the ability to assemble with beta 2. The reciprocal chimera containing Glu-Glu-Phe in the context of gamma 2 failed to assemble with beta 2. The last residue of this triplet is occupied by a leucine in all known mammalian gamma subunits except gamma 1 and appears to be a key determinant of the ability of a gamma subunit to assemble with beta 2. This locus maps to a region of predicted alpha-helical structure in the gamma subunit, likely to represent a point of physical contact with the beta subunit.
Highlights
The assembly of p and 'Y subtypes is selective
With respect to the 'Y subunits, there is only 38% sequence identity at the amino acid level between y1 [12] and y2 [13, 14], and little is known about the domain or domains that are responsible for their differential ability to assemble with
To study the subunit selectivity of the chimeras, they were cotransfected into COS-7 cells with either (31 or (32 . f3y assembly was determined by assessing the ability of these nonisoprenylated mutant y subunits to direct expression of f3 subunit to the soluble protein fraction [6, 7]
Summary
Nonisoprenylated 'Y constructs [6] This assay was recently used to study truncated and point-mutated {3 subunits [7], leading to the development of a model for f3'Y interaction in which the N terminus of {3 forms a coiled coil-like structure with 'Y· Pronin and Gautam [8] used a similar paradigm to study subunit specificity in f3'Y heterodimer formation. Using this and other approaches, several laboratories have demonstrated that y1 interacts with {3I but not with {32 , whereas both {3 subunits interact with y2 and y3 [8,9,10]. This putative {3-contact site lies just C-terminal to a proposed coiled coil region of the 'Y subunit
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