Podocytic Infolding Glomerulopathy in a Patient with Phospholipase A2 Receptor-Positive Membranous Nephropathy and Review of the Literature.

Podocytic infolding glomerulopathy (PIG) is a rare diagnosis based on characteristic histopathologic findings on renal biopsy and was proposed as a new entity about a decade ago. It is a type of podocytic injury, characterized by invagination of the podocyte cell membrane (cytoplasmic projections from podocytes) into the glomerular basement membrane. The presence of microspheres and/or microtubules on electron microscopy is the characteristic finding. PIG is most often associated with autoimmune conditions like systemic lupus erythematosus. We report pathologic findings typical of PIG in a patient with phospholipase A2 receptor antibody-positive membranous nephropathy. She was treated with rituximab and responded well with decrease in proteinuria to the sub-nephrotic range.

Heterotrimeric Gα12/13 proteins in kidney injury and disease.

Gα12 and Gα13 are ubiquitous members of the heterotrimeric guanine nucleotide-binding protein (G protein) family that play central and integrative roles in the regulation of signal transduction cascades within various cell types in the kidney. Gα12/Gα13 proteins enable the kidney to adapt to an ever-changing environment by transducing stimuli from cell surface receptors and accessory proteins to effector systems. Therefore, perturbations in Gα12/Gα13 levels or their activity can contribute to the pathogenesis of various renal diseases, including renal cancer. This review will highlight and discuss the complex and expanding roles of Gα12/Gα13 proteins on distinct renal pathologies, with emphasis on more recently reported findings. Deciphering how the different Gα12/Gα13 interaction networks participate in the onset and development of renal diseases may lead to the discovery of new therapeutic strategies.

Autosomal Recessive Alport Syndrome Unveiled by Pregnancy.

Alport syndrome is a hereditary disease affecting Type IV collagen characterized by hematuria, progressive renal failure, sensorineural hearing loss, and ocular abnormalities. Most cases are X-linked and involve the COL4A5 gene with a minority of patients having autosomal recessive mutations in the COL4A3 or COL4A4 genes encoding the α3(IV) or α4(IV) chain respectively. Here, we describe the case of a 31-year-old woman who presented during pregnancy with hematuria and proteinuria and was diagnosed with autosomal recessive Alport syndrome (ARAS) post-partum. Her biopsy was notable for findings of segmental glomerulosclerosis with some collapsing features, in addition to thin basement membranes and rare "splitting". Genetic testing identified 2 novel mutations in the COL4A4 gene: a truncating frame shift mutation c.3861delinsCTC and a missense mutation c.4708G>A (p.Glu1570Lys), both of which we assert to be pathogenic. She had normal full-term delivery without complications. This case has several unique features including the relatively mild disease phenotype and the findings of glomerular scarring with collapsing features on renal biopsy. The successful pregnancy outcome and her clinical presentation add to the growing body of evidence that ARAS can have a variable phenotype.

Physician and patient tools to improve chronic kidney disease care.

OBJECTIVES: To determine if electronic health record (EHR) tools and patient engagement can improve the quality of chronic kidney disease (CKD) care. STUDY DESIGN: Randomized controlled trial. METHODS: We enrolled 153 primary care physicians caring for 3947 high-risk and 3744 low-risk patients with stage III CKD across 13 ambulatory health centers in eastern Massachusetts. Intervention physicians received a set of electronic alerts during office visits recommending risk-appropriate CKD care. Patients of intervention physicians also received tailored educational mailings. For high-risk patients, we assessed for a visit with a nephrologist and prescription of an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) during the 12-month study period. For low-risk patients, we assessed for a urine microalbumin screening and prescription of an ACE inhibitor or ARB during the 12-month study period. RESULTS: Among high-risk patients, those in the intervention arm were significantly more likely to have an office visit with a nephrologist compared with those in the control arm (45% vs 34%; P <.001). Among low-risk patients, those in the intervention arm were significantly more likely than those in the control arm to have received urine microalbumin testing (45% vs 21%; P <.001). There was no difference between the intervention and control arms in rates of prescription of an ACE inhibitor or ARB in either the high-risk patient group (76% vs 79%; P = .17) or the low-risk patient group (64% vs 65%; P = .57). CONCLUSIONS: A combined program of EHR tools and patient engagement improved some areas of CKD care, but substantial gaps remain.

Gα12 is required for renal cystogenesis induced by Pkd1 inactivation.

Mutation of PKD1, encoding the protein polycystin-1 (PC1), is the main cause of autosomal dominant polycystic kidney disease (ADPKD). The signaling pathways downstream of PC1 in ADPKD are still not fully understood. Here, we provide genetic evidence for the necessity of Gα12 (encoded by Gna12, hereafter Gα12) for renal cystogenesis induced by Pkd1 knockout. There was no phenotype in mice with deletion of Gα12 (Gα12-/-). Polyinosine-polycytosine (pI:pC)-induced deletion of Pkd1 (Mx1Cre+Pkd1f/fGα12+/+) in 1-week-old mice resulted in multiple kidney cysts by 9 weeks, but the mice with double knockout of Pkd1 and Gα12 (Mx1Cre+Pkd1f/fGα12-/-) had no structural and functional abnormalities in the kidneys. These mice could survive more than one year without kidney abnormalities except multiple hepatic cysts in some mice, which indicates that the effect of Gα12 on cystogenesis is kidney specific. Furthermore, Pkd1 knockout promoted Gα12 activation, which subsequently decreased cell-matrix and cell-cell adhesion by affecting the function of focal adhesion and E-cadherin, respectively. Our results demonstrate that Gα12 is required for the development of kidney cysts induced by Pkd1 mutation in mouse ADPKD.

Kidney injury molecule-1 protects against Gα12 activation and tissue damage in renal ischemia-reperfusion injury.

Ischemic acute kidney injury is a serious untreatable condition. Activation of the G protein α12 (Gα12) subunit by reactive oxygen species is a major cause of tissue damage during renal ischemia-reperfusion injury. Kidney injury molecule-1 (KIM-1) is a transmembrane glycoprotein that is highly up-regulated during acute kidney injury, but the physiologic significance of this up-regulation is unclear. Here, we report for the first time that Kim-1 inhibits Gα12 activation and protects mice against renal ischemia-reperfusion injury. We reveal that Kim-1 physically interacts with and inhibits cellular Gα12 activation after inflammatory stimuli, including reactive oxygen species, by blocking GTP binding to Gα12. Compared with Kim-1(+/+) mice, Kim-1(-/-) mice exhibited greater Gα12 and downstream Src activation both in primary tubular epithelial cells after in vitro stimulation with H2O2 and in whole kidneys after unilateral renal artery clamping. Finally, we show that Kim-1-deficient mice had more severe kidney dysfunction and tissue damage after bilateral renal artery clamping, compared with wild-type mice. Our results suggest that KIM-1 is an endogenous protective mechanism against renal ischemia-reperfusion injury through inhibition of Gα12.

Polycystin-1 and Gα12 regulate the cleavage of E-cadherin in kidney epithelial cells.

Interaction of polycystin-1 (PC1) and Gα12 is important for development of kidney cysts in autosomal dominant polycystic kidney disease (ADPKD). The integrity of cell polarity and cell-cell adhesions (mainly E-cadherin-mediated adherens junction) is altered in the renal epithelial cells of ADPKD. However, the key signaling pathway for this alteration is not fully understood. Madin-Darby canine kidney (MDCK) cells maintain the normal integrity of epithelial cell polarity and adherens junctions. Here, we found that deletion of Pkd1 increased activation of Gα12, which then promoted the cystogenesis of MDCK cells. The morphology of these cells was altered after the activation of Gα12. By using liquid chromatography-mass spectrometry, we found several proteins that could be related this change in the extracellular milieu. E-cadherin was one of the most abundant peptides after active Gα12 was induced. Gα12 activation or Pkd1 deletion increased the shedding of E-cadherin, which was mediated via increased ADAM10 activity. The increased shedding of E-cadherin was blocked by knockdown of ADAM10 or specific ADAM10 inhibitor GI254023X. Pkd1 deletion or Gα12 activation also changed the distribution of E-cadherin in kidney epithelial cells and caused ß-catenin to shift from cell membrane to nucleus. Finally, ADAM10 inhibitor, GI254023X, blocked the cystogenesis induced by PC1 knockdown or Gα12 activation in renal epithelial cells. Our results demonstrate that the E-cadherin/ß-catenin signaling pathway is regulated by PC1 and Gα12 via ADAM10. Specific inhibition of this pathway, especially ADAM10 activity, could be a novel therapeutic regimen for ADPKD.

H2O2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury.

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify Gα12 activation by H(2)O(2) leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-MDCK) were subjected to ATP depletion/repletion and H(2)O(2)/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 completely protected cells from H(2)O(2)-stimulated barrier disruption, a response that rapidly occurred in control cells. H(2)O(2) activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H(2)O(2)-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H(2)O(2) led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.

Gα12 activation in podocytes leads to cumulative changes in glomerular collagen expression, proteinuria and glomerulosclerosis.

Glomerulosclerosis is a common pathological finding that often progresses to renal failure. The mechanisms of chronic kidney disease progression are not well defined, but may include activation of numerous vasoactive and inflammatory pathways. We hypothesized that podocytes are susceptible to filtered plasma components, including hormones and growth factors that stimulate signaling pathways leading to glomerulosclerosis. Gα12 couples to numerous G-protein-coupled receptors (GPCRs) and regulates multiple epithelial responses, including proliferation, apoptosis, permeability and the actin cytoskeleton. Herein, we report that genetic activation of Gα12 in podocytes leads to time-dependent increases in proteinuria and glomerulosclerosis. To mimic activation of Gα12 pathways, constitutively active Gα12 (QL) was conditionally expressed in podocytes using Nphs2-Cre and LacZ/floxed QLα12 transgenic mice. Some QLα12(LacZ+/Cre+) mice developed proteinuria at 4-6 months, and most were proteinuric by 12 months. Proteinuria increased with age, and by 12-14 months, many demonstrated glomerulosclerosis with ultrastructural changes, including foot process fusion and both mesangial and subendothelial deposits. QLα12(LacZ+/Cre+) mice showed no changes in podocyte number, apoptosis, proliferation or Rho/Src activation. Real-time PCR revealed no significant changes in Nphs1, Nphs2, Cd2ap or Trpc6 expression, but Col4a2 message was increased in younger and older mice, while Col4a5 was decreased in older mice. Confocal microscopy revealed disordered collagen IVα1/2 staining in older mice and loss of α5 without changes in other collagen IV subunits. Taken together, these studies suggest that Gα12 activation promotes glomerular injury without podocyte depletion through a novel mechanism regulating collagen (α)IV expression, and supports the notion that glomerular damage may accrue through persistent GPCR activation in podocytes.

The biology of epithelial cell tight junctions in the kidney.

Nearly 50 years have lapsed since the tight junction between epithelial cells was first identified by electron microscopy. The tight junction was once viewed as a static structure providing a barrier to paracellular movement and restricting proteins to the apical or basolateral membrane. Recent insights into the molecular composition of tight junctions reveal surprising complexity and dynamic regulation. Epithelia along the nephron exemplify a diversity of tight junctions that contribute to more than a 100-fold difference in permeability from the proximal tubule to the collecting duct. Tight junctions along the nephron form during kidney development and must reassemble after tubular injury. Hereditary diseases, animal models, and cell culture studies provide a variety of new perspectives on the function of tight junctions in health and disease.

Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1.

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α(2A)-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G(αq) but not G(α12). The compound inhibited thrombus formation in vivo following vascular injury with an IC(50) of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.

Primary care management of chronic kidney disease.

BACKGROUND: Chronic kidney disease (CKD) causes substantial morbidity and mortality; however, there are limited data to comprehensively assess quality of care in this area. OBJECTIVE: To assess quality of care for CKD according to patient risk and identify correlates of improved care delivery. DESIGN: Retrospective cohort. SETTING: Fifteen health centers within a multi-site group practice in eastern Massachusetts. PARTICIPANTS: 166 primary care physicians caring for 11,774 patients with stages 3 or 4 CKD defined as two estimated glomerular filtration rates (eGFR) between 15 and 60. MAIN MEASURES: Two measures of kidney disease monitoring, five measures of cardiovascular disease management, four measures of metabolic bone disease and anemia management, and one measure of drug safety were extracted from the electronic health record. Primary care recognition of CKD was assessed as a problem list diagnosis, and nephrology co-management was assessed as at least one visit with a nephrologist in the prior 12 months. KEY RESULTS: Overall, 46% of patients were high risk for death based on the presence of diabetes, proteinuria, or an eGFR <45. Seventy percent of patients lacked annual urine protein testing, 46% had a blood pressure ≥130/80 mmHg and 25% were not receiving appropriate angiotensin blockade. Appropriate screening for anemia was common (76%), while screening rates for metabolic bone disease were low. Use of potentially harmful drugs was common (26%). Primary care physician recognition and nephrology co-management were both associated with improved quality of care, though rates of both were low (24% and 10%, respectively). CONCLUSIONS: Significant deficiencies in the quality of CKD care exist. Opportunities for improvement include increasing physician recognition of CKD and improving collaborative care with nephrology.

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Gα12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of Gα12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Gα12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Gα12. We identified 3 NAAIRS substitutions in Gα12 that completely abrogated binding, and identified a previously described 74 amino acid Gαi/o binding domain in the PC1 C-terminus as necessary for Gα12 interaction. The functional consequences of uncoupling PC1/Gα12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Gα12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Gα12-stimulated apoptosis. Likewise, deletion of the Gα12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Gα12-stimulated apoptosis. Based on the crystal structure of Gα12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Gα12 signaling in ADPKD.

Regulation of integrin expression by Gα12: An additional potential mechanism modulating cell attachment.

Integrins regulate cell attachment and migration through interactions with specific proteins in the extra-cellular matrix. Heterotrimeric G proteins are essential signal transduction proteins that intersect with integrin signaling to regulate fundamental cellular behaviors. Although integrin and G protein signaling often act in concert, how these mechanisms interact in epithelial cells has not been extensively studied. We recently reported Gα12 regulation of epithelial cell attachment and migration on collagen-I through α2ß1 integrins (Kong et al. Mol Biol Cell 2009). Activated Gα12 inhibited α2ß1 integrin functions through an inside-out signaling mechanism that involved Rho, Src and protein phosphatases without affecting α2 or ß1 expression. Activated Gα12 prevented tubulogenesis in 3D-MDCK cell cultures and promoted the formation of cystic structures. Herein, we extend these findings to show Gα12-stimulated transcriptional changes in integrin expression that affect MDCK cell attachment. Based on results from a microarray with MDCK cells expressing constitutively active Gα12 (QLα12), we confirmed with real time PCR that expressing QLα12 led to a 4-fold inhibition of α6 mRNA expression. Cell surface expression and total α6 protein was reduced by FACS and immunofluorescence. QLα12 expressing MDCK cells also revealed less attachment to laminin-5, an α6 integrin ligand. Taken together, G proteins regulate integrins through canonical signaling pathways and potentially regulate integrin expression levels to modulate cellular responses in a variety of pathophysiologic conditions including polycystic kidney disease.

Polycystin-1 protein level determines activity of the Galpha12/JNK apoptosis pathway.

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Galpha(12). Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Galpha(12) stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352-24363). Herein, we confirm this pathway in Galpha(12)-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Galpha(12)-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Galpha(12), but not the related Galpha(13) subunit, and thrombin-stimulated MDCK cells led to increased interaction of Galpha(12) with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Galpha(12)/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.

G alpha 12 inhibits alpha2 beta1 integrin-mediated Madin-Darby canine kidney cell attachment and migration on collagen-I and blocks tubulogenesis.

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.

Heterotrimeric G proteins and apoptosis: intersecting signaling pathways leading to context dependent phenotypes.

Apoptosis, a programmed cell death mechanism, is a fundamental process during the normal development and somatic maintenance of all multicellular organisms and thus is highly conserved and tightly regulated through numerous signaling pathways. Apoptosis is of particular clinical importance as its dysregulation contributes significantly to numerous human diseases, primarily through changes in the expression and activation of key apoptotic regulators. Each of the four families of heterotrimeric G proteins (G(s), G(i/o), G(q/11) and G(12/13)) has been implicated in numerous cellular signaling processes, including proliferation, transformation, migration, differentiation, and apoptosis. Heterotrimeric G protein signaling is an important but not widely studied mechanism regulating apoptosis. G protein Signaling and Apoptosis broadly cover two large bodies of literature and share numerous signaling pathways. Examination of the intersection between these two areas is the focus of this review. Several studies have implicated signaling through each of the four heterotrimeric G protein families to regulate apoptosis within numerous disease contexts, but the mechanism(s) are not well defined. Each G protein family has been shown to stimulate and/or inhibit apoptosis in a context-dependent fashion through regulating numerous downstream effectors including the Bcl-2 family, NF-kappaB, PI3 Kinase, MAP Kinases, and small GTPases. These cell-type specific and G protein coupled receptor dependent effects have led to a complex body of literature of G protein regulation of apoptosis. Here, we review the literature and summarize apoptotic signaling through each of the four heterotrimeric G protein families (and the relevant G protein coupled receptors), and discuss limitations and future directions for research on regulating apoptosis through G protein coupled mechanisms. Continued investigation in this field is essential for the identification of important targets for pharmacological intervention in numerous diseases.

Galpha12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly.

The polarized functions of epithelia require an intact tight junction (TJ) to restrict paracellular movement and to separate membrane proteins into specific domains. TJs contain scaffolding, integral membrane and signaling proteins, but the mechanisms that regulate TJs and their assembly are not well defined. Galpha12 (GNA12) binds the TJ protein ZO-1 (TJP1), and Galpha12 activates Src to increase paracellular permeability via unknown mechanisms. Herein, we identify Src as a component of the TJ and find that recruitment of Hsp90 to activated Galpha12 is necessary for signaling. TJ integrity is disrupted by Galpha12-stimulated Src phosphorylation of ZO-1 and ZO-2 (TJP2); this phosphorylation leads to dissociation of occludin and claudin 1 from the ZO-1 protein complex. Inhibiting Hsp90 with geldanamycin blocks Galpha12-stimulated Src activation and phosphorylation, but does not affect protein levels or the Galpha12-ZO-1 interaction. Using the calcium-switch model of TJ assembly and GST-TPR (GST-fused TPR domain of PP5) pull-downs of activated Galpha12, we demonstrate that switching to normal calcium medium activates endogenous Galpha12 during TJ assembly. Thrombin increases permeability and delays TJ assembly by activating Galpha12, but not Galpha13, signaling pathways. These findings reveal an important role for Galpha12, Src and Hsp90 in regulating the TJ in established epithelia and during TJ assembly.