Regulation of signal transfer from beta1-adrenoceptor to adenylate cyclase by betagamma subunits in a reconstituted system

Abstract

The By subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, py subunits from these sources have been found to be fully interchangeable in terms of their interaction with a subunits of pertussistoxin-sensitive G proteins. In contrast, however, there are striking differences between these by subunits with regard to their ability to deactivate fluoride-stimulated G,. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of by subunits purified from bovine brain to vesicles containing P-receptor and G, results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin py was virtually without any effect. Likewise, by from bovine brain, but not transducin by, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, G,, C). Thus, the a subunit of G,, but not the a subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different Py subunits.It is now generally accepted that GTP-binding proteins play a universal role as receptor-effector couplers in hormonal and visual signal transduction. But whereas G, and Gi stimulate and inhibit adenylate cyclase, respectively, GI activates a retinal cGMP phosphodiesterase presumably by removing an inhibitory y subunit of the enzyme [l, 21. In addition, there are other G proteins, such as Go, G, and G, [3 -51. It is possible that these G proteins participate in other, not yet defined, receptor-effector coupling systems. All signaltransducing G proteins which have been characterized on a molecular basis have been found to be heterotrimers. The masses of the a subunits of the G proteins which bind and hydrolyze GTP range over 21 -52 kDa. All a subunits are substrates for ADP-ribosylation catalyzed by one or more bacterial toxin. Data obtained by molecular cloning have revealed that all a subunits studied so far differ structurally but that their homologies are great enough to consider them to belong to one class of related proteins [6]. The , ! 3 subunits of the G-protein heterotrimers isolated from different sources contain, with the exception of transducin, two chains with [8] indicate that the 35-kDa chain differs from the 36-kDa one. These data are corroborated by the existence of two genes for subunits [9]. Moreover, the y subunit of transducin seems to differ from all other known y subunits Until now, the information available is scarce on how the structural differences between the various by subunits may be related to functional differences. It was shown, for example, that transducin Py subunits and those from rabbit liver membranes can both support coupling of transducin a to lightactivated rhodopsin [12]. Moreover, it was shown by Cerione et al. [13] that transducin fly subunits can inhib...

Binding of α‐ and βγ‐subunits of G0 to β1‐adrenoceptor in sealed unilamellar lipid vesicles

“…Absorbance and protein determinations indicated that FITC was coupled to the purified B1-adrenoceptor in a molar ratio of approximately 1: 1. The ability of the fluorescent B-adrenoceptor to activate G, in the presence of I(-)isoproterenol and GTP[yS] was tested as described before [15] and was found to be like that of an unlabelled receptor preparation [18 b] (and Frohlich et al, unpublished results). The purity of the receptor preparation is shown in Fig.…”

“…GTPase activity was determined as described in [15]. Purified G-proteins in vesicles or in 0.1% Lubrol PX solution were incubated in 50 mM Hepes, 0.1 mM EDTA-Na2, 1 mM dithiothreitol and 0.2 mM MgC12 pH 7.8, with 1 pM [y-32P]GTP (3-6 cpm/fmol) for 20 min at 30°C.…”

Structural heterogeneity of membrane receptors and GTP‐binding proteins and its functional consequences for signal transduction

“…Moreover, the fly-complex alone attaches to the receptor (Im et al, 1988;Kurstjens et al, 1991) and appears to be part of the recognition site for G-protein -a-subunits. by-Subunits also contribute in a concentration-dependent manner both to the hormonal activation and to the deactivation of G,, (Hekman et al, 1987).…”

“…They are interchangeable among the a-subunits of the nonretinal G-proteins, G,, Gi, Go, but G,, can discriminate between transducin fly, and the fly complex resolved from the nonretinal G-proteins of the brain (Hekman et al, 1987;Casey et al, 1989). However, quantitative information is lacking.…”

Excursions in biophysics by a classical enzymologist

“…Moreover, when &-subunits purified from bovine brain were added to purified 0-adrenoceptor in lipid vesicles in the presence of G, also purified from turkey erythrocytes, the fly-subunits promoted the interaction of the /3-adrenoceptor with G, and increased its GTPase activity (Hekman et al, 1987). These observations became the starting point of a new line of research that I have followed ever since.…”