Reconstitution of beta-adrenergic receptor with components of adenylate cyclase.

Hekman, Mirko; D, Feder; Keenan, A. K.; Gal, Adiv; Klein, Helmut W.; Pfeuffer, Thomas; Levitzki, Alexander; Helmreich, Ernst

doi:10.1002/j.1460-2075.1984.tb02301.x

Cited by 66 publications

(29 citation statements)

References 32 publications

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“…[ Extractigel-D was from Pierce Chemical Co., and lauroyl sucrose was either prepared as described [14] or was a gift from Mitsubishi Co., Tokyo. BA 85 nitrocellulose filters were from Schleicher & Schiill, and ethyleneglycol monomethylether was from Merck, Darmstadt.…”

Section: Methodsmentioning

confidence: 99%

“…BA 85 nitrocellulose filters were from Schleicher & Schiill, and ethyleneglycol monomethylether was from Merck, Darmstadt. All other chemicals and biochemicals were of the highest grade commercially available and were from the same sources as described before [14,IS].…”

Section: Methodsmentioning

confidence: 99%

“…5. Aliquots (10 pl) were withdrawn and residual G, activity was measured by adenylate cyclase activation using the crude myocardial extract as source of the enzyme [14].…”

Section: The Binding Of [35s]gtp[ys]mentioning

confidence: 99%

“…The amount of 8-receptor in phospholipid vesicles or in digitonin solution was assessed by binding with 240 pM [ '251]iodocyanopindolol or 50 nM [3H]dihydroalprenolo1, respectively, as described in [14]. Adenylate cyclase activities were measured either with crude myocardial extract or with purified cyclase according to the procedure described in [14].…”

Section: Other Assaysmentioning

confidence: 99%

“…Adenylate cyclase activities were measured either with crude myocardial extract or with purified cyclase according to the procedure described in [14]. 13'P]cAMP formed was isolated by the method of Salomon et al [25].…”

Section: Other Assaysmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…5. Aliquots (10 pl) were withdrawn and residual G, activity was measured by adenylate cyclase activation using the crude myocardial extract as source of the enzyme [14].…”

Section: The Binding Of [35s]gtp[ys]mentioning

confidence: 99%

Section: Other Assaysmentioning

confidence: 99%

Section: Other Assaysmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 1987

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Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

Pfeuffer¹,

Mollner²,

Pfeuffer³

1985

The EMBO Journal

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100

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The non-stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000-fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin-agarose. The final product appears as one major band (mol. wt. 115 000) on SDS-polyacrylamide gels. Further identification was achieved by affinity cross-linking using Gs (stimulatory GTPbinding protein) that was [32P]ADP-ribosylated by choleratoxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, -270 000 and higher. The distribution of these products was dependent on the concentration of cross-linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photoaffinity cross-linking with 4-azidobenzoyl-[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP-analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin-Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.

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

D¹,

Im²,

Klein³

et al. 1986

The EMBO Journal

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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)

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Reconstitution of beta-adrenergic receptor with components of adenylate cyclase.

Cited by 66 publications

References 32 publications

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

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

Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

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

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