Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs.alpha.

Sutkowski, Elizabeth McHugh; Tang, Wei-Jen; Broome, Christopher W.; Robbins, J B; Seamon, Kenneth B.

doi:10.1021/bi00209a017

Cited by 148 publications

(81 citation statements)

References 31 publications

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“…2, all three forms of AC were stimulated by G s␣ and forskolin. As described by McHugh-Sutkowski et al (27) for the fulllength ACV, when G s␣ and forskolin were added together, the activity of the full-length enzyme in Sf9 cells was only modestly increased over the sum of the stimulation observed with forskolin and G s␣ alone (Fig. 2C).…”

Section: Resultssupporting

confidence: 74%

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Scholich

Barbier

Mullenix

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Type V adenylyl cyclase (ACV) belongs to the family of Ca 2+ - inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the α subunit of the stimulatory G protein G s (G sα ) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the α subunit of the inhibitory G protein G i (G iα ) inhibited all forms of the enzyme, indicating that the regions for G sα and G iα interaction are preserved in the soluble forms. Ca 2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G sα and forskolin obliterated the Ca 2+ -mediated inhibition of the full-length and C1-C2 forms of ACV. In 45 Ca 2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca 2+ . Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45 Ca 2+ . To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G sα and G iα is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca 2+ and inhibition of enzyme activity.

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Section: Resultssupporting

confidence: 74%

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Scholich

Barbier

Mullenix

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…First, several ACs are expressed in the cells assayed here and they have different sensitivities to forskolin. If AC7 is less sensitive to forskolin than other ACs as has been reported for AC2 (39), any synergism may get lost in the large signal from other ACs in the cells. Second, even though an AC can be activated by forskolin, the activation state of the protein appears to have a different conformation than when it is activated by G s (40 -42).…”

Section: Discussionmentioning

confidence: 87%

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

Jiang

Collins

Davis

et al. 2008

Journal of Biological Chemistry

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Regulation of intracellular cAMP by multiple pathways enables differential function of this ubiquitous second messenger in a context-dependent manner. Modulation of G s -stimulated intracellular cAMP has long been known to be modulated by the G i and G q /Ca 2؉ pathways. Recently, the G 13 pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the G s and G 13 pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the ␣ subunit of either G 12 or G 13 . Our results demonstrate that AC7 is a specific downstream effector of the G 12/13 pathway.

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“…7) Therefore, the increased level of free G s a subunits due to expression of G s protein-coupled receptors with constitutive activity would enhance the responses to forskolin. Alewijnse et al 8) demonstrated that constitutive activity of G s protein-coupled receptors, such as histamine H 2 receptor and thyrotropin receptor, contribute to the enhancement of both basal and forskolin-induced cAMP production in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Influence of Receptor Number on the cAMP Response to Forskolin in Chinese Hamster Ovary Cells Transfected with Human .BETA.2-Adrenoceptor

Nakahara

Maruko

Sakamoto

et al. 2004

Biological & Pharmaceutical Bulletin

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b 2 -Adrenoceptor agonists are potent bronchodilators and are widely used in the treatment of asthma. However, the continuous or repeated exposure to these drugs reduces the bronchodilation mediated by b 2 -adrenoceptors. The mechanism of the reduced responsiveness to b 2 -adrenoceptor agonists has been studied extensively. Nowadays, the uncoupling of b 2 -adrenoceptor to G s protein and down-regulation of the receptor are recognized as the key process in the phenomenon.1) Thus, the number of receptors expressed in the airway smooth muscle cell appears to be important in determining the response to stimulation of b 2 -adrenoceptor.Previous reports demonstrated that increasing expression of b 2 -adrenoceptors in stably transfected cells increased basal cAMP levels and the potency of isoproterenol in stimulating cAMP formation.2-4) However, the influence of receptor number of b 2 -adrenoceptor on cAMP response evoked by the direct activation of adenylyl cyclase has not been clearly addressed. The purpose of this study, therefore, was to examine how density of b 2 -adrenoceptor affects the forskolin-induced cAMP accumulation in Chinese hamster ovary (CHO) cells transfected with human b 2 -adrenoceptor. MATERIALS AND METHODSThe cDNA encoding human b 2 -adrenoceptor was kindly provided by Dr. R. J. Lefkowitz (Duke University). For expression, the human b 2 adrenoceptor cDNA was inserted into pEF/myc/cyto (Invitrogen Corp., Carlsbad, Calif., U.S.A.) at the NcoI and SalI restriction sites. Chinese hamster ovary cells (CHO-K1 cells) were transfected with pEF/myc/cyto-b 2 using lipofectAMINE (Life Technologies, Grand Island, NY, U.S.A.) according to the manufacturer's instructions. Clones were isolated by selection with G418 (1 mg/ml) and were screened for b 2 -adrenoceptor expression in a [3 H]CGP 12177 radioligand binding assay as described below. Culture medium was Ham's F-12 Nutrient mixture supplemented with 10% of fetal bovine serum.Membrane Preparation Stably expressed CHO cells were grown to ca. 80% confluence at 37°C in 5% CO 2 /95% humidified air. The cells were washed, and scraped into icecold lysis buffer, and homogenized at 20000 rpm for 60 s with the tissue homogenizer. The lysis buffer consisted of 10 mM Tris-HCl (pH 7.4) containing 5 mM EDTA, 5 mM EGTA, 2.5 mg/ml pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride and 1 mM leupeptin. Membranes were pelleted at 45000ϫg for 30 min and resuspended in 10 mM HepesNaOH (pH 7.4) to give a final concentration of 1 mg/ml and stored at Ϫ80°C until the receptor binding assay was carried out.Radioligand Receptor Binding Assay Radioligand receptor binding studies were conducted in reaction buffer containing 50 mM Tris-HCl (pH 7.4) and 10 mM MgCl 2 at 37°C for 60 min using 50 mg of membrane protein. For saturation isotherms, membranes were incubated with various concentrations (0.2-6.8 nM) of [ 3 H]CGP 12177 (37 Ci/mmol, NEN Life Science Products, Inc., Boston, MA, U.S.A.) with or without propranolol (10 mM) for 60 min. Reactions were terminated with rapid filtration tho...

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Regulation of forskolin interactions with type I, II, V, and VI adenylyl cyclases by Gs.alpha.

Cited by 148 publications

References 31 publications

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Regulation of cAMP Responses by the G12/13 Pathway Converges on Adenylyl Cyclase VII

Influence of Receptor Number on the cAMP Response to Forskolin in Chinese Hamster Ovary Cells Transfected with Human .BETA.2-Adrenoceptor

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