Studies on the interaction of α subunits of GTP‐binding proteins with βγ dimers

Abstract

The interaction of several preparations of purified p;, dimers with two types of guanosinenucleotide-binding-regulatory-(G)-protein a subunits, a recombinant bvai3, made in Sf9 Spuduptern frugiperdu cells by the baculovirus (bv) expression system, and as, either purified from human erythrocyte G,-type GTP-binding protein, and activated by NaF/A1C13, or unpurified as found in a natural membrane, were studied. The By dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of p subunits that, upon electrophoresis, migrated as two bands with approximate M , of 35000 and 36000, as well as distinct complements of at least two ; J subunits each. When tested for their ability to rccombinc at submaximal concentrations with bv,xi3, ROS, brain, hRBC and placental py dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS /?y dimers were tested for their ability to promote deactivation of G,, brain and placental by dimers were equipotent and at least 10-fold more potent than that of KOS p1~ dimers; likewise, brain by and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenolactivated adenylyl cyclase, while ROS by dimers were less potent when assayed at the same concentration. The possibility that different CI subunits may distinguish subsets of By dimers from a single cell was investigated by analyzing the by composition of three G proteins, G,, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an a-subunitspecific difference in fly composition was found. These findings suggests that, in most cells, a subunits interact indistinctly with a common pool of / +$ dimers. However, since at least one By preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of py dimers may exhibit selectivity for the a subunits they interact with.One of the characteristics of the trimeric signal transducing heterotrimetric GTP-binding proteins of apy subunit composition (G proteins) is that they can be activated by GTP analogs such as guanosine 5'-ly-thio]triphosphate (GTP[yS]) to give two reaction products: a complex of the guanine nucleotide with the Y subunit and a free By dimer.Although not yet proven unequivocally, the dissociation reaction is believed to occur also in membranes containing GTP, undcr thc catalytic effect of ligand-activated receptors [l , 21. It has been speculated [2] and proposed [3, 41 that both a subunits and fly dimers may regulate effector functions under physiologic conditions. Such a role has indeed been firmly established for a subunits, which act at picomolar concentrations in several

Purification of the G-protein G13 from rat brain membranes

“…The amino-terminal region was identified to be important for interaction of Ga,-, Gas-Ga.-subunits with flydimers [32][33][34]. Furthermore, amino-terminal truncation of recombinant Gae3 reduced its affinity for ,y-dimers [23]. [40,41].…”

“…Sucrose-density-gradient centrifugation was performed essentially as described previously [22,23]. Linear sucrose gradients [32P]NAD' was synthesized from [a-32P]ATP as described by Cassel and Pfeuffer [24].…”

Signal transduction by a nondissociable heterotrimeric yeast G protein

“…Indeed, several different combinations of G ␤␥ subunits have been shown to interact with the same G ␣ in vitro (25). The sequence of the S. cerevisiae genome revealed only two G ␣ homologs (GPA1 and GPA2), but eight candidate ␤ genes and three candidate ␥ genes (26).…”

Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin

“…Numerous biochemical (29,31,47,48) and mutational (49,50) studies have implicated the amino terminus of G protein ␣ subunits in the interaction with ␤␥ subunits. The resolution of the crystal structure of heterotrimeric G t (14) has confirmed that the amino-terminal helix of the ␣ t subunit is involved in forming a binding site for the ␤ t subunit.…”