Identification of RGS2 and Type V Adenylyl Cyclase Interaction Sites

Salim, Samina; Sinnarajah, Srikumar; Kehrl, John H.; Dessauer, Carmen W.

doi:10.1074/jbc.m210663200

Cited by 131 publications

(113 citation statements)

References 43 publications

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“…17). In addition, the N-terminus of RGS2 can bind to and inhibit type V adenylyl cyclase (Salim et al 2003). GPCRs themselves have become partners for RGS2 interaction, as shown recently for the α1A adrenoceptor, potentially by interaction of the N-terminal domain of RGS2 with the third intracellular loop of GPCRs mediated by the protein spinophilin in a Ca 2+ sensitive fashion ( Fig.…”

Section: Genetic Variants Of the Regulator Of G Protein Signalling (Rmentioning

confidence: 72%

Genetics of arterial hypertension and hypotension

Rosskopf

Schürks

Rimmbach

et al. 2007

Naunyn-Schmied Arch Pharmacol

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Human hypertension affects affects more than 20% of the adult population in industrialized countries, and it is implicated in millions of deaths worldwide each year from stroke, heart failure and ischemic heart disease. Available evidence suggests a major genetic impact on blood pressure regulation. Studies in monogenic hypertension revealed that renal salt and volume regulation systems are predominantly involved in the genesis of these disorders. Mutations here affect the synthesis of mineralocorticoids, the function of the mineralocorticoid receptor, epithelial sodium channels and their regulation by a new class of kinases, termed WNK kinases. It has been learned from monogenic hypotension that almost all ion transporters involved in the renal uptake of Na + have a major impact on blood pressure regulation. For essential hypertension as a complex disease, many candidate genes have been analysed. These include components of the reninangiotensin-aldosterone system, adducin, β-adrenoceptors, G protein subunits, regulators of G protein signalling (RGS) proteins, Rho kinases and G protein receptor kinases. At present, the individual impact of common polymorphisms in these genes on the observed blood pressure variation, on risk for stroke and as predictors of antihypertensive responses remains small and clinically irrelevant. Nevertheless, these studies have greatly augmented our knowledge on the regulation of renal functions, cellular signal transduction and the integration of both. Together, this provides the basis for the identification of novel drug targets and, hopefully, innovative antihypertensive drugs.

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Section: Genetic Variants Of the Regulator Of G Protein Signalling (Rmentioning

confidence: 72%

Genetics of arterial hypertension and hypotension

Rosskopf

Schürks

Rimmbach

et al. 2007

Naunyn-Schmied Arch Pharmacol

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“…␤1␥2-H6 was purified from Sf9 cells (32). Hexa-histidine-and GST-tagged proteins were purified on Ni-NTA agarose (Qiagen) and glutathione resin (Amersham) in buffers lacking detergents as previously described (31). Proteins were dialyzed into buffer containing 50 mM Hepes pH 8.0, 100 mM NaCl, 5% glycerol, 2 mM DTT, and 1 mM EDTA, and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

“…Protein Purification. G␣s-H 6 was expressed in E. coli and activated with [ 35 S]GTP␥S as previously described (31). ␤1␥2-H6 was purified from Sf9 cells (32).…”

Section: Methodsmentioning

confidence: 99%

The A-kinase anchoring protein Yotiao binds and regulates adenylyl cyclase in brain

Piggott

Bauman

Scott

et al. 2008

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

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132

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A-kinase anchoring proteins (AKAPs) influence the spatial and temporal regulation of cAMP signaling events. Anchoring of PKA in proximity to certain adenylyl cyclase (AC) isoforms is thought to enhance the phosphorylation dependent termination of cAMP synthesis. Using a combination of immunoprecipitation and enzymological approaches, we show that the plasma membrane targeted anchoring protein AKAP9/Yotiao displays unique specificity for interaction and the regulation of a variety of AC isoforms. Yotiao inhibits AC 2 and 3, but has no effect on AC 1 or 9, serving purely as a scaffold for these latter isoforms. Thus, Yotiao represents an inhibitor of AC2. The N terminus of AC2 (AC2-NT), which binds directly to amino acids 808 -957 of Yotiao, mediates this interaction. Additionally, AC2-NT and Yotiao (808 -957) are able to effectively inhibit the association of AC2 with Yotiao and, thus, reverse the inhibition of AC2 by Yotiao in membranes. Finally, disruption of Yotiao-AC interactions gives rise to a 40% increase in brain AC activity, indicating that this anchoring protein functions to directly regulate cAMP production in the brain. 2). This family of proteins functions to target PKA and other cAMP effector proteins to specific regions of the cell allowing for increased signaling specificity. Several members of this diverse protein family bind other receptors, channels, or enzymes to form tightly regulated signaling modules, facilitating PKA interactions with its downstream effectors. In addition, these signaling modules often constitute feedback loops between kinases and phosphatases or recruit phosphodiesterases to terminate the cAMP signal (3).Our labs previously identified a complex containing AKAP79/ 150 and type 5 adenylyl cyclase (AC), that facilitates the spatiotemporal organization of cAMP signaling (4). AKAP79 inhibits AC5 activity, while providing a mechanism for feedback inhibition via PKA phosphorylation of anchored AC5. However, with Ϸ30 gene families of AKAPs identified thus far, and nine mammalian isoforms of AC, it begs the question, does AKAP scaffolding of AC and PKA represent a general paradigm for cAMP signaling? The AKAP Yotiao represents an ideal test case to begin to address this question. Yotiao is a splice variant of the AKAP9 gene and is present on the plasma membrane (5, 6). In addition to PKA, Yotiao binds protein phosphatase 1 (PP1), NMDA receptors, the heart potassium channel subunit KCNQ1, and the IP 3 R1 (5, 7-9). Interestingly, AKAP anchored PKA phosphorylation of the NMDA receptor, KCNQ1, and IP 3 R1 is necessary for each effector's normal function (8-11). For example, disruption of the KCNQ1 association with Yotiao gives rise to long QT syndrome, a type of heart arrhythmia that can be fatal (12). The requirement for a tight coupling between PKA and KCNQ1, and other effectors suggests that cAMP production must also be tightly regulated, perhaps as even part of this complex.We now show that Yotiao is associated with brain and heart adenylyl cyclases. Yotiao displays specificity am...

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“…Protein was expressed in Escherichia coli and purified under non-denaturing conditions using glutathione affinity resin essentially as described (33), except that NaCl concentration was increased to 250 mM in the lysis buffer. The protein was dialyzed overnight in 20 mM Hepes, 1 mM EDTA, 2 mM DTT, 100 mM NaCl, and 5% glycerol.…”

Section: Methodsmentioning

confidence: 99%

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

Gao

Sadana

Dessauer

et al. 2007

Journal of Biological Chemistry

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In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein ␤2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the G␤ 1 subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that G␤␥ subunits enhance the activity of these enzymes provided either G␣ s or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of G␤␥ subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by G␤␥ subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous G␤␥ subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G i was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, G␤␥ subunits both in vitro and in intact cells. Moreover, G␤␥ subunits derived from a source(s) other than G i are necessary for the full activation of hACVI by isoproterenol in intact cells.

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Identification of RGS2 and Type V Adenylyl Cyclase Interaction Sites

Cited by 131 publications

References 43 publications

Genetics of arterial hypertension and hypotension

Genetics of arterial hypertension and hypotension

The A-kinase anchoring protein Yotiao binds and regulates adenylyl cyclase in brain

Conditional Stimulation of Type V and VI Adenylyl Cyclases by G Protein βγ Subunits

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