Critical cysteines in the functional interaction of adenylyl cyclase isoform 6 with Gαs

Bhagirath, Anjali Y.; Bhatia, Vikram; Medapati, Manoj Reddy; Singh, Nisha; Hinton, Martha; Chelikani, Prashen; Dakshinamurti, Shyamala

doi:10.1096/fba.2021-00073

Cited by 4 publications

(6 citation statements)

References 68 publications

(88 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is known that small or local perturbations can produce significant RMSD changes. The magnitude of RMSD we report following a single residue substitution mutation is in line with what we have previously obtained from single-point mutation analyses [ 44 ]. Similar magnitude RMSD changes are described by others after the substitution of hydrophobic alanine for a neutral (e.g., threonine) or hydrophilic (e.g., asparagine) residue [ 54 ], and from single point mutations occurring spontaneously in the SARS-CoV2 protease that result in altered molecular dynamics [ 55 ].…”

Section: Discussionsupporting

confidence: 90%

“…Despite the increase in expression, we observed that the activity of the AC6 isoform is significantly inhibited under hypoxia [ 17 ]. Hypoxia may impair AC6 interaction with its key upstream activator Gαs [ 44 ] due to nitrosylation of AC6 at its interface with Gαs [ 22 , 45 ]. Similarly, we found nitrosyl group donor CSNO treatment decreases AC activity only in the AC6 isoform.…”

Section: Discussionmentioning

confidence: 99%

“…All 9 isoforms of ACs share some sequence similarities, mainly at the catalytic domain site, as well as regulatory patterns. Most previous published AC structures are chimera-based approaches [ 44 ]. Almost two decades ago, molecular structures of AC were reported to exist in three conformational states of AC: (1) catalytically inactive IIC 2 , (2) Gαs and forskolin bound IIC 2 complexed with VC 1 , and (3) Gαs and forskolin bound IIC 2 complexed with VC 1 , adenosine analog, and pyrophosphate [ 47 ].…”

Section: Discussionmentioning

confidence: 99%

“…This confirms our earlier report of decreased AC activity when pulmonary myocytes predominantly expressing AC6 were exposed to hypoxia [ 17 ], and shows that hypoxia sensitivity is a unique feature of AC6. We have previously investigated cysteines involved in the AC6- Gαs interaction; mutation of cysteine 1004 inhibits basal AC activity and cAMP generation by altering the interaction of AC6 with Gαs versus Gαi [ 44 ]. In this study, we used a similar approach focusing on FSK-dependent AC activity.…”

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Characterization of Adenylyl Cyclase Isoform 6 Residues Interacting with Forskolin

Bhatia

Maghsoudi

Hinton

et al. 2023

Biology

Self Cite

View full text Add to dashboard Cite

Background: The adenylyl cyclase (AC) pathway, crucial for pulmonary vasodilation, is inhibited by hypoxia. Forskolin (FSK) binds allosterically to AC, stimulating ATP catalysis. As AC6 is the primary AC isoform in the pulmonary artery, selective reactivation of AC6 could provide targeted reinstatement of hypoxic AC activity. This requires elucidation of the FSK binding site in AC6. Methods: HEK293T cells stably overexpressing AC 5, 6, or 7 were incubated in normoxia (21% O2) or hypoxia (10% O2) or exposed to s-nitrosocysteine (CSNO). AC activity was measured using terbium norfloxacin assay; AC6 structure built by homology modeling; ligand docking to examine FSK-interacting amino acids; roles of selected residues determined by site-directed mutagenesis; FSK-dependent cAMP generation measured in wild-type and FSK-site mutants by biosensor-based live cell assay. Results: Only AC6 is inhibited by hypoxia and nitrosylation. Homology modeling and docking revealed residues T500, N503, and S1035 interacting with FSK. Mutation of T500, N503, or S1035 decreased FSK-stimulated AC activity. FSK site mutants were not further inhibited by hypoxia or CSNO; however, mutation of any of these residues prevented AC6 activation by FSK following hypoxia or CSNO treatment. Conclusions: FSK-interacting amino acids are not involved in the hypoxic inhibition mechanism. This study provides direction to design FSK derivatives for selective activation of hypoxic AC6.

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Characterization of Adenylyl Cyclase Isoform 6 Residues Interacting with Forskolin

Bhatia

Maghsoudi

Hinton

et al. 2023

Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Gαi association with membrane receptors is known to increase when it is nitrosylated ( Chao et al, 2021 ). We also reported in PASMC a switch from Gαs-to Gαi-signalling in a nitrosylating environment ( Bhagirath et al, 2022 ). Receptor G protein coupling dynamics deserve further examination in presence of ROS/RNS in order to elucidate the mechanisms for NO augmentation of hypoxic Ca 2+ mobilization.…”

Section: Discussionmentioning

confidence: 87%

Nitric oxide augments signaling for contraction in hypoxic pulmonary arterial smooth muscle—Implications for hypoxic pulmonary hypertension

Hinton

Thliveris²,

Hatch³

et al. 2023

Front. Physiol.

Self Cite

View full text Add to dashboard Cite

Introduction: Hypoxic persistent pulmonary hypertension in the newborn (PPHN) is usually treated with oxygen and inhaled nitric oxide (NO), both pulmonary arterial relaxants. But treatment failure with NO occurs in 25% of cases. We previously demonstrated that 72 h exposure to hypoxia, modeling PPHN, sensitized pulmonary artery smooth muscle cells (PASMC) to the contractile agonist thromboxane and inhibited relaxant adenylyl cyclase (AC) activity.Methods: In this study, we examined the effects of sodium nitroprusside (SNP), as NO donor, on the thromboxane-mediated contraction and NO-independent relaxation pathways and on reactive oxygen species (ROS) accumulation in PASMC. In addition, we examined the effect of the peroxynitrite scavenger 5,10,15,20-Tetrakis (4-sulfonatophenyl)porphyrinato Iron (III) (FeTPPS) on these processes.Results: Exposure of PASMC to 72 h hypoxia increased total intracellular ROS compared to normoxic control cells and this was mitigated by treatment of cells with either SNP or FeTPPS. Total protein nitrosylation was increased in hypoxic PASMC compared to controls. Both normoxic and hypoxic cells treated with SNP exhibited increased total protein nitrosylation and intracellular nitrite; this was reduced by treatment with FeTPPS. While cell viability and mitochondrial number were unchanged by hypoxia, mitochondrial activity was decreased compared to controls; addition of FeTPPS did not alter this. Basal and maximal mitochondrial metabolism and ATP turnover were reduced in hypoxic PASMC compared to controls. Hypoxic PASMC had higher basal Ca2+, and a heightened peak Ca2+ response to thromboxane challenge compared to controls. Addition of SNP further elevated the peak Ca2+ response, while addition of FeTPPS brought peak Ca2+ response down to control levels. AC mediated relaxation was impaired in hypoxic PASMC compared to controls but was normalized following treatment with FeTPPS. Addition of SNP inhibited adenylyl cyclase activity in both normoxic and hypoxic PASMC. Moreover, addition of the Ca2+ chelator BAPTA improved AC activity, but the effect was minimal.Discussion: We conclude that NO independently augments contraction and inhibits relaxation pathways in hypoxic PASMC, in part by a mechanism involving nitrogen radical formation and protein nitrosylation. These observations may partially explain impaired effectiveness of NO when treating hypoxic pulmonary hypertension.

show abstract