Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

D, Feder; Im, Mie‐Jae; Klein, Helmut W.; Hekman, Mirko; Holzhöfer, Andreas; Dees, Christian; Levitzki, Alexander; Helmreich, Ernst; Pfeuffer, Thomas

doi:10.1002/j.1460-2075.1986.tb04390.x

Cited by 93 publications

(31 citation statements)

References 29 publications

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“…A considerable effort was made to detect a change in the affinity of the ,&lAR on coreconstitution with &-subunits into lipid vesicles using methods established in this laboratory [7,18]. However, the reconstituted turkey PrAR did not respond well to interaction with either nonactivated or activated forms of G, with a shift from a high-affinity to low-affinity state in the presence A 8…”

Section: Resultsmentioning

confidence: 99%

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Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Im¹,

Holzhöfer²,

Böttinger³

et al. 1988

FEBS Letters

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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.

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Section: Resultsmentioning

confidence: 99%

“…The Pi-adrenoceptors from turkey erythrocyte membranes were purified according to published procedures [18,19]. G, and Gb from turkey erythrocytes were purified as described by Northup et al [IO].…”

Section: Methodsmentioning

confidence: 99%

Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Im¹,

Holzhöfer²,

Böttinger³

et al. 1988

FEBS Letters

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“…The P1-adrenergic receptor from turkey erythrocyte membranes was purified by affinity chromatography following a published protocol [15] which was modified as follows. Briefly, turkey erythrocyte membranes (5 mg protein/ml) were solubilized in 10 mM Tris/HCl and 90 mM NaCl buffer (pH 7.6) in the presence of 0.4% lauroyl sucrose and protease inhibitors for 40 min at 4"C, followed by centrifugation at 40000 x g for 40 min.…”

Section: Purification Of Proteinsmentioning

confidence: 99%

“…About 50% of G, incorporated into vesicles were hormone-dependently activated. Phospholipid vesicles containing purified catalytic subunit of adenylate cyclase were prepared and assayed as described [15].…”

Section: Reconstitutionmentioning

confidence: 99%

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

et al. 1987

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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...

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“…One of the signal transduction systems studied in great detail involves the activation of adenylyl cyclase by p-adrenergic receptors. In this system, all the components have been identified, purified and reconstituted in a synthetic membrane [6,7]. The finding that S49 cyc-cells have a mutation that lacks the G,a subunit but *Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

Human‐Xenopus chimeras of G_sα reveal a new region important for its activation of adenylyl cyclase

et al. 1994

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G proteins are heterotrimeric GTPases that play a key role in signal transduction. The a subunit of G, bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X laevis cDNA clones that apparently code for G,a subunits are 92% identical to those found in the short form of human G,a. Despite this high homology, the X laevis G,a clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc-membranes in contrast with human G,a similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human G,a. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus G,a. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPys and isoproterenol. The results obtained here clearly indicate that the Ga sequence that extends from amino acid 70 to 140, is important for the functionality of human G,a in activating adenylyl cyclase.

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Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

Cited by 93 publications

References 29 publications

Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

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

Human‐Xenopus chimeras of G_sα reveal a new region important for its activation of adenylyl cyclase

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Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components.

Cited by 93 publications

References 29 publications

Interactions of pure βγ‐subunits of G‐proteins with purified β1‐adrenoceptor

Interactions of pure βγ‐subunits of G‐proteins with purified β1‐adrenoceptor

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

Human‐Xenopus chimeras of Gsα reveal a new region important for its activation of adenylyl cyclase

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Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Interactions of pure βγ‐subunits of G‐proteins with purified β₁‐adrenoceptor

Human‐Xenopus chimeras of G_sα reveal a new region important for its activation of adenylyl cyclase