Gamma Subunit Types Research Articles

A G Protein γ Subunit-specific Peptide Inhibits Muscarinic Receptor Signaling

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein gamma5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the gamma5 peptide inhibits modulation of Ca(2+) current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein gamma subunit family are significantly less effective. The gamma5 peptide has no effect on Ca(2+) current modulation by the alpha2-adrenergic and somatostatin receptors. In addition, the gamma5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein gamma subunit types in signal transduction, most likely at the receptor-G protein interface.

The Formation or the Reduction of a Disulfide Bridge on the γ Subunit of Chloroplast ATP Synthase Affects the Inhibitory Effect of the ε Subunit

We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.

Differential Ability to Form the G Protein βγ Complex among Members of the β and γ Subunit Families

We have determined the relative abilities of several members of the G protein beta and gamma subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian beta1 and gamma3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of beta and gamma subunit types varies widely. Third, the reporter activity evoked by a particular combination of beta and gamma subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid betagamma complex formed in the cell as determined by immunoprecipitation. These results suggest that different beta and gamma subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all gamma subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins.

Immunoaffinity purification of gamma-aminobutyric acidA (GABAA) receptors containing gamma 1-subunits. Evidence for the presence of a single type of gamma-subunit in GABAA receptors.

Two polyclonal antibodies were raised against the gamma 1-subunit of gamma-aminobutyric acidA (GABAA) receptors. One of these antibodies could be used for the immunopurification of GABAA receptors containing gamma 1-subunits. These receptors exhibited [3H]muscimol and [3H]flunitrazepam binding, and their benzodiazepine binding properties were dramatically different from those of receptors precipitated by anti-gamma 2- or anti-gamma 3-antibodies. Western blot analysis of the immunopurified GABAA receptors indicated that the gamma 1-subunit exhibits an apparent molecular mass of 45-51 kDa. Furthermore, in addition to gamma 1-subunits, beta 2/3-, alpha 1-, alpha 2-, alpha 3-, and alpha 5-subunits could be detected in immunoaffinity column eluates from total brain. In contrast, gamma 2- or gamma 3-subunits could not be identified in GABAA receptors immunopurified by anti-gamma 1-antibodies. Similarly, gamma 1- and gamma 3-subunits or gamma 1- and gamma 2-subunits could not be identified in GABAA receptors purified by anti-gamma 2- or anti-gamma 3-antibodies, respectively. These data seem to indicate that GABAA receptors contain only a single type of gamma-subunit.

Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein.

Interaction with a receptor is the first step in the process of signal transduction by heterotrimeric (alpha beta gamma) G proteins. We have examined the role of the G protein gamma subunit in interaction between a receptor (rhodopsin) and a G protein, transducin (Gt). We have obtained recombinant beta gamma complexes containing the same beta subunit but three different gamma subunit types by expressing them in the baculovirus/insect cell system. We show that the different recombinant beta gamma complexes interact equally well with the alpha subunit of transducin (alpha t) but only the gamma subunit specific to rod photoreceptors (gamma 1) is able to support interaction of alpha t with rhodopsin. This indicates a direct role for the G protein gamma subunits, which are a family of proteins with diverse structures, in conferring specificity to receptor-G protein interaction.

Interaction between G-protein beta and gamma subunit types is selective.

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association.