Purification and characterization of five different α subunits of guanine‐nucleotide‐binding proteins in bovine brain membranes

Abstract

We have purified five different a subunits of guanine-nucleotide-binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5'-[y-thio]triphosphate (GTP[yS]). All the purified a subunits were interacted with py subunits and served as a substrate for pertussin-catalyzed ADP-ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various CI subunits of G proteins, three of them were identified as M~-~, M~-~ and M~-~, and the other two were classified into a, type. One of the a,-type proteins was the most abundant in the brain membranes (termed CI,), and the other (a,2) appeared to differ from a, in its proteolytic digestion data. The physiological properties of these purified GTP[yS]-bound a subunits towards adenylate cyclase and atrial muscarinic K + channels were studied. The nucleotide-bound forms of ai-2, M~-~ and a,2 inhibited the adenylate cyclase activity of S49 cyc-membranes which had been reconstituted with GTP[yS]-treated G,; this inhibition appeared to be mainly competitive with the activated G,, ai-l having the most potent inhibitory activity among them. GTP[yS]-bound a,, however, could not inhibit the G,-stimulated activity at all. On the other hand, all the GTP[yS]-bound a subunits activated atrial muscarinic K + channels, accompanied by a lag time, at picomolar concentrations. The fly subunits resolved from G proteins also activated the K + channels without a lag time at nanomolar concentration. The maximum activation by the by subunits appeared to be more potent than that by any of the a subunits. These results suggest that CI and fly subunits might activate the K + channels by mechanisms different from each other.Guanine-nucleotide-binding proteins (G proteins) are a family of signal-coupling proteins that play key roles in many hormonal and sensory transduction processes in eukaryotes (for review see [l -31). G proteins, which have a common heterotrimetric structure consisting of an a, p and y subunit, carry signals from various membrane-bound receptors to effectors such as enzymes or ion channels. The ci subunits have a high-affinity binding site specific for GTP (or GDP) and a site for NAD-dependent ADP-ribosylation which is catalyzed by bacterial toxins such as pertussin (also termed isletactivating protein or IAP) and/or cholera toxin. Functions of G proteins as signal transducers are profoundly affected by these toxin-induced modifications of the M subunits. Thus, pertussin and cholera toxin have been widely used as tools to identify these G proteins and to analyze their functions. G proteins serving as the substrate of pertussin were initially classified into Gi and Go according to their distribution in the types of cells, besides two GI that were only present in retinal rod or cone. Further studies of the molecular cloning of a subunit gene [4] and cDNAs [5 -71 indicated the existence of at least three genes for the M subunit of Gi, namely the ai-z and ai-3. In contrast, only a single type of the a subunit of Go ...

Purification of the G-protein G13 from rat brain membranes

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PIP 3 inhibition of RGS protein and its reversal by Ca 2+ /calmodulin mediate voltage-dependent control of the G protein cycle in a cardiac K + channel

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Specific roles of Gi protein family members revealed by dissecting SST5 coupling in human pituitary cells

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