Proteomic cellular signatures of kinase inhibitor-induced cardiotoxicity.

Drug Toxicity Signature Generation Center (DToxS) at the Icahn School of Medicine at Mount Sinai is one of the centers for the NIH Library of Integrated Network-Based Cellular Signatures (LINCS) program. Its key aim is to generate proteomic and transcriptomic signatures that can predict cardiotoxic adverse effects of kinase inhibitors approved by the Food and Drug Administration. Towards this goal, high throughput shotgun proteomics experiments (308 cell line/drug combinations +64 control lysates) have been conducted. Using computational network analyses, these proteomic data can be integrated with transcriptomic signatures, generated in tandem, to identify cellular signatures of cardiotoxicity that may predict kinase inhibitor-induced toxicity and enable possible mitigation. Both raw and processed proteomics data have passed several quality control steps and been made publicly available on the PRIDE database. This broad protein kinase inhibitor-stimulated human cardiomyocyte proteomic data and signature set is valuable for prediction of drug toxicities.

Kai­Xin­San suppresses matrix metalloproteinases and myocardial apoptosis in rats with myocardial infarction and depression.

Depression is often triggered by prolonged exposure to psychosocial stressors and associated with coronary heart disease (CHD). Matrix metalloproteinases (MMPs) are involved in the pathogenesis of various emotional and cardiovascular disorders. The purpose of this study was to investigate whether Kai­Xin­San (KXS), which may terminate the signaling of MMPs, exerts antidepressant­like and cardioprotective effects in a myocardial infarction (MI) plus depression rat model. Rats were randomly assigned to five groups: A normal control (control group), a celisc­injection of isopropyl adrenaline group (ISO group), depression (depression group), an ISO + depression (depression + ISO group), and an ISO + depression group treated with intragastric administration of 1,785 mg/kg KXS (KXS group). Behavioral changes, echocardiography, biochemical index, matrix metalloproteinase (MMP) and apoptosis­related proteins were assessed. Compared with the depression + ISO group, KXS significantly improved stress­induced alterations of behavioral parameters and protected the heart by enlarging the left ventricular (LV) fractional shortening (FS) and LV ejection fraction (EF). Moreover, KXS significantly attenuated ISO + depression­induced MMP­2 and MMP­9 expression at the mRNA and protein level and decreased TIMP in the heart compared to the complex model group. Myocardial apoptosis was significantly attenuated by KXS by regulating the Bcl­2/Bax axis. These results indicated that MI comorbid with depression may damage the MMP balance in the central and peripheral system, and KXS may have a direct anti­depressive and cardio­protective effect by regulating the level of MMPs and associated myocardial apoptosis. It is promising to further explore the clinical potential of KXS for the therapy or prevention of MI plus depression comorbidity disease.

Genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer identified by systems therapeutics analyses.

The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop transcriptomic signatures for specific drugs. We analyzed lung adenocarcinoma patient data from The Cancer Genome Atlas and identified a subset of patients in which xanthine dehydrogenase (XDH) expression correlated with decreased survival. We tested allopurinol, an FDA-approved drug that inhibits XDH, on human non-small-cell lung cancer (NSCLC) cell lines obtained from the Broad Institute Cancer Cell Line Encyclopedia and identified sensitive and resistant cell lines. We utilized the transcriptomic profiles of these cell lines to identify six-gene signatures for allopurinol-sensitive and allopurinol-resistant cell lines. Transcriptomic networks identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 was verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinol-sensitive NSCLC cell lines and four allopurinol-resistant cell lines susceptible to combination therapy. We searched the transcriptomic data from a library of patient-derived NSCLC tumors from the Jackson Laboratory to identify tumors that would be predicted to be sensitive to allopurinol or allopurinol + CEP-33779 treatment. Patient-derived tumors showed the predicted drug sensitivity in vivo. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

Cell shape information is transduced through tension-independent mechanisms.

The shape of a cell within tissues can represent the history of chemical and physical signals that it encounters, but can information from cell shape regulate cellular phenotype independently? Using optimal control theory to constrain reaction-diffusion schemes that are dependent on different surface-to-volume relationships, we find that information from cell shape can be resolved from mechanical signals. We used microfabricated 3-D biomimetic chips to validate predictions that shape-sensing occurs in a tension-independent manner through integrin ß3 signaling pathway in human kidney podocytes and smooth muscle cells. Differential proteomics and functional ablation assays indicate that integrin ß3 is critical in transduction of shape signals through ezrin-radixin-moesin (ERM) family. We used experimentally determined diffusion coefficients and experimentally validated simulations to show that shape sensing is an emergent cellular property enabled by multiple molecular characteristics of integrin ß3. We conclude that 3-D cell shape information, transduced through tension-independent mechanisms, can regulate phenotype.

A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction.

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young's modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.

Fragility of foot process morphology in kidney podocytes arises from chaotic spatial propagation of cytoskeletal instability.

Kidney podocytes' function depends on fingerlike projections (foot processes) that interdigitate with those from neighboring cells to form the glomerular filtration barrier. The integrity of the barrier depends on spatial control of dynamics of actin cytoskeleton in the foot processes. We determined how imbalances in regulation of actin cytoskeletal dynamics could result in pathological morphology. We obtained 3-D electron microscopy images of podocytes and used quantitative features to build dynamical models to investigate how regulation of actin dynamics within foot processes controls local morphology. We find that imbalances in regulation of actin bundling lead to chaotic spatial patterns that could impair the foot process morphology. Simulation results are consistent with experimental observations for cytoskeletal reconfiguration through dysregulated RhoA or Rac1, and they predict compensatory mechanisms for biochemical stability. We conclude that podocyte morphology, optimized for filtration, is intrinsically fragile, whereby local transient biochemical imbalances may lead to permanent morphological changes associated with pathophysiology.

Enhanced Antitumor Activity of Cetuximab in Combination with the Jak Inhibitor CYT387 against Non-Small-Cell Lung Cancer with Various Genotypes.

Cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is effective in the treatment of non-small-cell lung cancers (NSCLCs). However, resistance to EGFR inhibitors limits its effectiveness. In this study, we investigated the effectiveness of Jak-2 inhibitor, CYT387, in combination with cetuximab. Xenograft animal models were administered with cetuximab or CYT387 or their combination. It was observed that NSCLC cells exhibited enormous differences in responses to cetuximab; cell lines were more intrinsically resistant to cetuximab. In resistant cell lines (H1975 and H1650), the efficacy of cetuximab was increased when combined with CYT387, whereas CYT387 alone in low doses exhibited little effect on NSCLC cell proliferation. In addition, the antitumor activity of cetuximab was increased in H1975 resistant model in spite of low efficacy of cetuximab treatment alone in. Jak/STAT signaling was suppressed effectively by the combination of cetuximab and CYT387. In summary, our findings indicated that CYT387 has a potent indirect antitumor activity, and it is also synergistic in its activity in combination with cetuximab against NSCLC tumors, especially with cetuximab intrinsic-resistance tumors. These indications were mediated via Janus kinase (Jak)-signal transducer and transcription (STAT) pathway activator. Our results strongly and consistently supported the potential synergism of CYT387 as Jak inhibitor for anti-NSCLC therapy with EGFR-targeting agents.

Determination of monoamine neurotransmitters in human urine by carrier-mediated liquid-phase microextraction based on solidification of stripping phase.

A novel method was developed for the analysis of monoamine neurotransmitters (MNTs) in human urine by carrier-mediated liquid-phase microextraction based on solidification of stripping phase method (CM-LPME-SSP) coupled with high performance liquid chromatography-electrochemical detector (HPLC-ECD). By adding an appropriate carrier in organic phase, simultaneous extraction of hydrophilic analytes, MNTs, with high enrichment factors (22.6-36.1 folds) and excellent sample cleanup was achieved. A new strategy, solidifying the aqueous stripping phase in the back-extraction process, was developed to facilitate the collection of the stripping phase as small as a few microliters. Combined with HPLC-ECD analysis, the linear ranges of the established method were 0.015-2.0 µg/mL for NE, E, DA, and 0.020-2.0 µg/mL for 5-HT. The limits of detection and quantification were in the range of 5.5-10.8 ng/mL and 15-20 ng/mL, respectively. The relative recoveries were in the range of 87-108%, with intraday and interday relative standard deviations lower than 13%. This method was successfully applied to analysis of MNTs in real urine.

Determination of catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high-performance liquid chromatography and electrochemical detection.

A new method was developed for the simultaneous determination of three catecholamines in urine using aminophenylboronic acid functionalized magnetic nanoparticles extraction followed by high-performance liquid chromatography with electrochemical detection. Novel aminophenylboronic acid functionalized magnetic nanoparticles were prepared by multi-step covalent modification, and characterized by transmission electron microscopy, Fourier-transformed infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometry. With the help of the high affinity between the boronate and cis-diol group, the particles were used for the highly selective separation and enrichment of three major catecholamines, norepinephrine, epinephrine, and dopamine. Effects of the pH of the feed solution, the extraction time, the composition of the buffer solution, the amount of the magnetic particles, the elution conditions, and the recycling of aminophenylboronic acid functionalized magnetic nanoparticles were explored. Under the optimized conditions, 13-17-fold enrichment factors were obtained. The linear ranges were 0.01-2.0 µg/mL for the studied analytes. The limits of detection and quantification were in the range of 2.0-7.9 and 6.7-26.3 ng/mL, respectively. The relative recoveries were in the range of 92-108%, with intraday and interday relative standard deviations lower than 6.8%. This method was successfully applied to analysis of catecholamines in real urine.

Inhibition of the JAK/STAT pathway with ruxolitinib overcomes cisplatin resistance in non-small-cell lung cancer NSCLC.

This study was aimed to elucidate the roles of inhibition of related JAK/STAT pathways in regulating cytotoxicity induced by cisplatin in non-small-cell lung cancer (NSCLC) cell. We treated five non-small-cell lung cancer cell lines with cisplatin alone or with cisplatin and Jak2 inhibitor (ruxolitinib) and assessed cell viability, expression of Jak2 and STAT3 and cell apoptosis. We also investigated the effect of combination treatment inhibited tumor xenograft growth in two human NSCLC xenograft models bearing the cisplatin resistant (H1299) and sensitive (A549) cells. Different cell lines with different genetic background showed half-maximal inhibitory concentrations (IC50) of cisplatin from 4.66 to 68.28 µmol/L. They could be divided into cisplatin intrinsic resistant and cisplatin sensitive cell lines. In cisplatin-resistant cells with higher Jak2 and STAT3 expression, cisplatin and ruxolitinib combination dramatically suppressed the cell growth, down-regulated the expression of phosphorylated STAT3 and induced cleaved caspase-3 expression. Moreover combination with cisplatin and ruxolitinib also significantly inhibited the growth of resistant cell H1299, A549/DDP and H2347 in soft agar model. Finally, combination group significant inhibited the tumor growth and induced the caspase-3 expression compared with either single agent alone (P < 0.05) on the resistant cell xenografts model. The present study indicates that further study is warranted to determine the effectiveness of combination treatment with cisplatin and Jak2/stat3 pathway inhibitor for platinum-resistant NSCLC.

Antidepressant and neuroprotective effect of the Chinese herb kaixinsan against lentiviral shRNA knockdown brain-derived neurotrophic factor-induced injury in vitro and in vivo.

Depression has been associated with reduced expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. Previous studies have demonstrated that the herbal medicine formula, 'kaixinsan' (KXS), could ameliorate the severity of depression and increase cAMP response element-binding protein expression. There is direct evidence suggesting that the reduction of the BDNF protein in specific brain sites can provoke depressive-like behaviour or affect neurogenesis in vivo. However, the biological mechanisms between the antidepressant and neuroprotective effect of KXS and the alterations in BDNF levels in in vivo and in vitro models remain unclear. Using BDNF knockdown mediated by lentiviral vectors (LV-shBDNF-3) transfected with primary hippocampal neurons and following injection into the dentate gyrus of the hippocampus, it was demonstrated that a reduction in BDNF expression affects cell viability and animal behaviours associated with depression. During treatment with KXS after the lentiviral shRNA silencing of BDNF in cell and animal, cell viability, body weight, the sucrose preference test (SPT), the open field test (OFT) the Morris Water Maze (MWM) task and BDNF expression were measured. KXS attenuated LV-shBDNF-3-induced cell death in primary hippocampal neurons and also improved the sucrose intake in SPT, ambulatory response in OFT and learning ability in MWM against LV-shBDNF-3-induced depressive-like syndromes. Moreover, immunoblot analysis confirmed that KXS could reverse LV-shBDNF-induced BDNF reduction either in vitro or in vivo. These findings provide substantial evidence for supporting a neurotrophic hypothesis of depression and specify BDNF targets for potential antidepressant interventions. Moreover, the antagonism between LV-shRNA BDNF knockdown and KXS may depend on multiple compounds with synergistic mechanisms that modulate the different signal transduction networks directly or indirectly, increasing BDNF expression and exerting its neuroprotective and antidepressant-like effects.

Interconnected network motifs control podocyte morphology and kidney function.

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a ß-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Systems pharmacology of adverse event mitigation by drug combinations.

Drugs are designed for therapy, but medication-related adverse events are common, and risk/benefit analysis is critical for determining clinical use. Rosiglitazone, an efficacious antidiabetic drug, is associated with increased myocardial infarctions (MIs), thus limiting its usage. Because diabetic patients are often prescribed multiple drugs, we searched for usage of a second drug ("drug B") in the Food and Drug Administration's Adverse Event Reporting System (FAERS) that could mitigate the risk of rosiglitazone ("drug A")-associated MI. In FAERS, rosiglitazone usage is associated with increased occurrence of MI, but its combination with exenatide significantly reduces rosiglitazone-associated MI. Clinical data from the Mount Sinai Data Warehouse support the observations from FAERS. Analysis for confounding factors using logistic regression showed that they were not responsible for the observed effect. Using cell biological networks, we predicted that the mitigating effect of exenatide on rosiglitazone-associated MI could occur through clotting regulation. Data we obtained from the db/db mouse model agreed with the network prediction. To determine whether polypharmacology could generally be a basis for adverse event mitigation, we analyzed the FAERS database for other drug combinations wherein drug B reduced serious adverse events reported with drug A usage such as anaphylactic shock and suicidality. This analysis revealed 19,133 combinations that could be further studied. We conclude that this type of crowdsourced approach of using databases like FAERS can help to identify drugs that could potentially be repurposed for mitigation of serious adverse events.

Limiting cardiac ischemic injury by pharmacological augmentation of macrophage migration inhibitory factor-AMP-activated protein kinase signal transduction.

BACKGROUND: Macrophage migration inhibitory factor (MIF) exerts a protective effect on ischemic myocardium by activating AMP-activated protein kinase (AMPK). Small molecules that increase the affinity of MIF for its receptor have been recently designed, and we hypothesized that such agonists may enhance AMPK activation and limit ischemic tissue injury. METHODS AND RESULTS: Treatment of cardiomyocytes with the candidate MIF agonist, MIF20, augmented AMPK phosphorylation, increased by 50% the surface localization of glucose transporter, and enhanced by 25% cellular glucose uptake in comparison with MIF alone. In mouse hearts perfused with MIF20 before no-flow ischemia and reperfusion, postischemic left ventricular function improved commensurately with an increase in cardiac MIF-AMPK activation and an augmentation in myocardial glucose uptake. By contrast, small-molecule MIF agonism was not effective in cells or tissues genetically deficient in MIF or the MIF receptor, verifying the specificity of MIF20 for MIF-dependent AMPK signaling. The protective effect of MIF20 also was evident in an in vivo regional ischemia model. Mice treated with MIF20 followed by left coronary artery occlusion and reperfusion showed a significant reduction in infarcted myocardium. CONCLUSIONS: These data support the pharmacological utility of small-molecule MIF agonists in enhancing AMPK activation and reducing cardiac ischemic injury.

A potential peptide therapeutic derived from the juxtamembrane domain of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645-662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed in vivo in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.

A Plasmodium-encoded cytokine suppresses T-cell immunity during malaria.

The inability to acquire protective immunity against Plasmodia is the chief obstacle to malaria control, and inadequate T-cell responses may facilitate persistent blood-stage infection. Malaria is characterized by a highly inflammatory cytokine milieu, and the lack of effective protection against infection suggests that memory T cells are not adequately formed or maintained. Using a genetically targeted strain of Plasmodium berghei, we observed that the Plasmodium ortholog of macrophage migration inhibitory factor enhanced inflammatory cytokine production and also induced antigen-experienced CD4 T cells to develop into short-lived effector cells rather than memory precursor cells. The short-lived effector CD4 T cells were more susceptible to Bcl-2-associated apoptosis, resulting in decreased CD4 T-cell recall responses against challenge infections. These findings indicate that Plasmodia actively interfere with the development of immunological memory and may account for the evolutionary conservation of parasite macrophage migration inhibitory factor orthologs.

Novel retinoic acid receptor alpha agonists for treatment of kidney disease.

Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARß, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN.

The D-dopachrome tautomerase (DDT) gene product is a cytokine and functional homolog of macrophage migration inhibitory factor (MIF).

Macrophage migration inhibitory factor (MIF) is a pivotal regulator of the immune response. Neutralization or genetic deletion of MIF does not completely abrogate activation responses, however, and deletion of the MIF receptor, CD74, produces a more pronounced phenotype than MIF deficiency. We hypothesized that these observations may be explained by a second MIF-like ligand, and we considered a probable candidate to be the protein encoded by the homologous, D-dopachrome tautomerase (D-DT) gene. We show that recombinant D-DT protein binds CD74 with high affinity, leading to activation of ERK1/2 MAP kinase and downstream proinflammatory pathways. Circulating D-DT levels correlate with disease severity in sepsis or malignancy, and the specific immunoneutralization of D-DT protects mice from lethal endotoxemia by reducing the expression of downstream effector cytokines. These data indicate that D-DT is a MIF-like cytokine with an overlapping spectrum of activities that are important for our understanding of MIF-dependent physiology and pathology.

Identification of new Gßγ interaction sites in adenylyl cyclase 2.

The role of Gßγ in adenylyl cyclase (AC) signaling is complicated due to its role as a conditional activator (AC2, AC4 and AC7) and an inhibitor (AC1, AC3 and AC8). AC2 is stimulated by Gα(s) and if Gßγ is present the stimulation is synergistic. The precise mechanism of this synergistic activation is still not known. In order to further elucidate the role of Gßγ in AC2 activation by Gα(s), peptides derived from the C1 domains of AC2 were synthesized and the ability of the various peptides to regulate AC2 function was tested. Our results identify two new Gßγ-binding sites in the AC2 C1 domain, AC2 C1a 339-360 and AC2 C1b 578-602 that are involved with stimulation of AC2 by Gßγ. These two regions are different from the previously described QEHA motif in the C2 domain of AC2. Further, the recently discovered PFAHL motif was confirmed to bind and to be involved with stimulation of AC2 by Gßγ. These functional studies indicate that multiple regions of AC2 are involved in the interaction with Gßγ.