In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1‐adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone‐dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP‐binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1‐adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl‐polyoxyethylene (octyl‐POE) was almost as potent as lauroyl‐sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol‐induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min‐1, whereas kon with rabbit liver GS was 0.6 min‐1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane‐bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4‐fold), stimulation by isoproterenol/GTP was modest (1.3‐ to 1.6‐fold).(ABSTRACT TRUNCATED AT 250 WORDS)

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...

The role of the βγ‐subunits in the interaction of G‐proteins was examined with β1‐adrenoceptors purified from turkey erythrocytes and pure βγ‐subunits prepared from turkey erythrocytes and bovine brain. On a non‐denaturing polyacrylamide gel, the mobility of βγ‐subunits was increased when incubated with β1‐adrenoceptor and the β1‐adrenergic agonist l‐(−)‐isoproterenol, whereas on incubation with the antagonist l‐alprenolol the mobility was unchanged. Furthermore, the β1‐adrenoceptor was retarded on a Sephadex G‐50 column equilibrated with βγ‐subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated β1‐adrenoceptors with isolated βγ‐subunits of G‐proteins.

Heterologous desensitization of turkey erythrocyte β‐adrenoceptors correlates with receptor phosphorylation and impaired receptor‐Gs coupling, as assessed by fusion of purified desensitized receptors with X. laevis erythrocytes [(1984) Science 225, 837‐840]. We have purified β‐receptors from desensitized and untreated turkey erythrocytes and have compared the abilities of these two receptors to couple with pure turkey erythrocyte Gs in a reconstituted system. Functional receptor‐Gs coupling was assessed by measuring hormone‐dependent (i) Gs, activation by GTPγS and (ii) GTPase activity. While in membranes prepared from desensitized cells, receptor‐Gs, coupling was clearly reduced, this effect was absent when coupling of purified desensitized receptor was measured. We conclude that covalent modification by phosphorylation does not fully explain the functional uncoupling at the membrane level.

The properties of a reconstituted signal transmission chain using purified beta 1-adrenoceptor (R), G-protein subunits (G) and adenylate cyclase (C) in lipid vesicles are described. This assay system was used to test beta, gamma-subunits of different origin with respect to their effects on R X G and R X G X C coupling and on the functional properties of GS alpha and Gi alpha. The findings reported here point to large differences in the efficacy of beta, gamma-subunits from different sources assessed by deactivation of [ALF4]-activated rabbit liver GS and pertussis toxin-catalyzed ADP-ribosylation of bovine neutrophil G alpha. This is explained by differences in the interaction domains of the interacting subunits. Furthermore, the sensitivity of R X G and R X G X C coupling to inhibition by beta, gamma-subunits was greater than the effects of beta, gamma-subunits on hormonally activated GTPase activity of GS. One of the consequences of differential inhibition of R X G X C coupling is an amplified response of the signal transmission chain to hormonal activation. This is in agreement with observations reported by Cerione et al.