TLR2 regulates hair follicle cycle and regeneration via BMP signaling.

The etiology of hair loss remains enigmatic, and current remedies remain inadequate. Transcriptome analysis of aging hair follicles uncovered changes in immune pathways, including Toll-like receptors (TLRs). Our findings demonstrate that the maintenance of hair follicle homeostasis and the regeneration capacity after damage depend on TLR2 in hair follicle stem cells (HFSCs). In healthy hair follicles, TLR2 is expressed in a cycle-dependent manner and governs HFSCs activation by countering inhibitory BMP signaling. Hair follicles in aging and obesity exhibit a decrease in both TLR2 and its endogenous ligand carboxyethylpyrrole (CEP), a metabolite of polyunsaturated fatty acids. Administration of CEP stimulates hair regeneration through a TLR2-dependent mechanism. These results establish a novel connection between TLR2-mediated innate immunity and HFSC activation, which is pivotal to hair follicle health and the prevention of hair loss and provide new avenues for therapeutic intervention.

Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.

Timely Wound Healing Is Dependent on Endothelial but Not on Hair Follicle Stem Cell Toll-Like Receptor 2 Signaling.

As a part of innate immunity, toll-like receptor 2 (TLR2) plays an important function in most defensive responses of the organism, including but not limited to infections. Cutaneous injury, one of the most common challenges for mammals, mobilizes a number of cell types, including epithelial, immune, and vascular cells, for timely tissue repair. However, in contrast to immune cells, little is known about TLR2 function on nonimmune cells during skin regeneration. In this study, we used two tissue-specific conditional Tlr2-knockout mouse lines to address the effects of TLR2 in endothelial and hair follicle stem cells (HFSCs) on cutaneous wound healing. The loss of TLR2 on endothelial cells diminishes their ability to migrate, sprout, and proliferate in response to specific TLR2 ligands and also reduces the secretion of key proangiogenic factors. Lack of TLR2 on endothelial cells prolongs wound healing owing to diminished angiogenesis. TLR2 is expressed in key structures of hair follicles, including HFSCs, secondary hair germ, and dermal papilla. Despite the prominent role of HFSCs in skin regeneration, excision of TLR2 from HFSCs has no effects on their proliferation or wound healing potential. Our study shows that timely tissue regeneration after skin injury is dependent on endothelial TLR2 for robust angiogenesis, whereas HFSC TLR2 is dispensable.

Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability.

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

Rab5-dependent autophagosome closure by ESCRT.

In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17-Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17-Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

Integrin-Kindlin3 requirements for microglial motility in vivo are distinct from those for macrophages.

Microglia play a critical role in the development and homeostasis of the CNS. While mobilization of microglia is critical for a number of pathologies, understanding of the mechanisms of their migration in vivo is limited and often based on similarities to macrophages. Kindlin3 deficiency as well as Kindlin3 mutations of integrin-binding sites abolish both integrin inside-out and outside-in signaling in microglia, thereby resulting in severe deficiencies in cell adhesion, polarization, and migration in vitro, which are similar to the defects observed in macrophages. In contrast, while Kindlin3 mutations impaired macrophage mobilization in vivo, they had no effect either on the population of microglia in the CNS during development or on mobilization of microglia and subsequent microgliosis in a model of multiple sclerosis. At the same time, acute microglial response to laser-induced injury was impaired by the lack of Kindlin3-integrin interactions. Based on 2-photon imaging of microglia in the brain, Kindlin3 is required for elongation of microglial processes toward the injury site and formation of phagosomes in response to brain injury. Thus, while Kindlin3 deficiency in human subjects is not expected to diminish the presence of microglia within CNS, it might delay the recovery process after injury, thereby exacerbating its complications.

Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site.

The epithelial brush-border Na(+)/H(+) exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2',7'-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser(554), Ser(607), and Ser(663), equivalent to mouse Ser(552), Ser(605), and Ser(659)), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser(663) phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser(554), Ser(607), and Ser(663), all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser(663). The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.

Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells.

The intestinal brush border Na(+)/H(+) exchanger NHE3 is tightly regulated through changes in its endocytosis and exocytosis. Myosin VI, a minus-end-directed actin motor, has been implicated in endocytosis at the inter-microvillar cleft and during vesicle remodeling in the terminal web. Here, we asked whether myosin VI also regulates NHE3 movement down the microvillus. The basal NHE3 activity and its surface amount, determined by fluorometry of the ratiometric pH indicator BCECF and biotinylation assays, respectively, were increased in myosin-VI-knockdown (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immunoelectron microscopy results showed that NHE3 was preferentially localized in the basal half of control microvilli but in the distal half in myosin VI KD cells. Treatment with dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis. However, NHE3 had an intermediate distribution along the microvillus (between that in myosin VI KD and untreated cells) in dynasore-treated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells, and suggest that myosin VI also moves NHE3 down the microvillus.

NHERF2 protein mobility rate is determined by a unique C-terminal domain that is also necessary for its regulation of NHE3 protein in OK cells.

Na(+)/H(+) exchanger regulatory factor (NHERF) proteins are a family of PSD-95/Discs-large/ZO-1 (PDZ)-scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins that contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2, and NHERF3 were all shown by fluorescence recovery after photobleaching/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, although different among the three, were all of the same magnitude as that of the transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, and NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a nonconserved region along with the ezrin, radixin, moesin (ERM) binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by lysophosphatidic acid of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.

NHERF2 is necessary for basal activity, second messenger inhibition, and LPA stimulation of NHE3 in mouse distal ileum.

To test the hypothesis that Na(+)/H(+) exchanger (NHE) regulatory factor 2 (NHERF2) is necessary for multiple aspects of acute regulation of NHE3 in intact mouse small intestine, distal ileal NHE3 activity was determined using two-photon microscopy/SNARF-4F in a NHERF2-null mouse model. The NHERF2-null mouse ileum had shorter villi, deeper crypts, and decreased epithelial cell number. Basal rates of NHE3 activity were reduced in NHERF2-null mice, which was associated with a reduced percentage of NHE3 in the apical domain and an increase in intracellular NHE3 amount but no change in total level of NHE3 protein. cAMP, cGMP, and elevated Ca(2+) due to apical exposure to UTP all inhibited NHE3 activity in wild-type mouse ileum but not in NHERF2-null mice, while inhibition by hyperosmolarity occurred normally. The cAMP-increased phosphorylation of NHE3 at aa 552; levels of PKAIIα and cGMP-dependent protein kinase II (cGKII); and elevation of Ca(2+) were similar in wild-type and NHERF2-null mouse ileum. Luminal lysophosphatidic acid (LPA) stimulated NHE3 in wild-type but not in NHERF2-null ileum. In conclusion, 1) there are subtle structural abnormalities in the small intestine of NHERF2-null mouse which include fewer villus epithelial cells; 2) the decreased basal NHE3 activity and reduced brush border NHE3 amount in NHERF2-null mice show that NHERF2 is necessary for normal basal trafficking or retention of NHE3 in the apical domain; 3) hyperosmolar inhibition of NHE3 occurs similarly in wild-type and NHERF2-null ileum, demonstrating that some inhibitory mechanisms of NHE3 are not NHERF2 dependent; 4) cAMP inhibition of NHE3 is NHERF2 dependent at a step downstream of cAMP/PKAII phosphorylation of NHE3 at aa 552; 5) cGMP- and UTP-induced inhibition of NHE3 are NHERF2 dependent at steps beyond cGKII and the UTP-induced increase of intracellular Ca(2+); and 6) LPA stimulation of NHE3 is also NHERF2 dependent.

D-glucose acts via sodium/glucose cotransporter 1 to increase NHE3 in mouse jejunal brush border by a Na+/H+ exchange regulatory factor 2-dependent process.

BACKGROUND & AIMS: Oral rehydration solutions reduce diarrhea-associated mortality. Stimulated sodium absorption by these solutions is mediated by the Na(+)/H(+) hydrogen exchanger NHE3 and is increased by Na(+)-glucose co-transport in vitro, but the mechanisms of this up-regulated process are only partially understood. METHODS: Intracellular pH was measured in jejunal enterocytes of wild-type mice and mice with disrupted Na+/H+ exchange regulatory co-factor 2 (NHERF2-/- mice) by multiphoton microscopy. Diarrhea was induced by cholera toxin. Caco-2BBe cells that express NHE3 and the sodium/glucose cotransporter 1 (SGLT1) were studied by fluorometry, before and after siRNA-mediated knockdown of NHERF1 or NHERF2. NHE3 distribution was assessed by cell-surface biotinylation and confocal microscopy. Brush-border mobility was determined by fluorescence recovery after photobleaching and confocal microscopy. RESULTS: The nonmetabolized SGLT1 substrate α-methyl-D-Glu (α-MD-G) activated jejunal NHE3; this process required Akt and NHERF2. α-MD-G normalized NHE3 activity after cholera toxin-induced diarrhea. α-MD-G-stimulated jejunal NHE3 activity was defective in NHERF2-/- mice and cells with NHERF2 knockdown, but occurred normally with NHERF1 knockdown; was associated with increased NHE3 surface expression in Caco-2 cells, which also was NHERF2-dependent; was associated with dissociation of NHE3 from NHERF2 and an increase in the NHE3 mobile fraction from the brush border; and was accompanied by a NHERF2 ezrin-radixin-moesin-binding domain-dependent increase in co-precipitation of ezrin with NHE3. CONCLUSIONS: SGLT1-mediated Na-glucose co-transport stimulates NHE3 activity in vivo by an Akt- and NHERF2-dependent signaling pathway. It is associated with increased brush-border NHE3 and association between ezrin and NHE3. Activation of NHE3 corrects cholera toxin-induced defects in Na absorption and might contribute to the efficacy of oral rehydration solutions.

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3.

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

Shiga toxin is transported into the nucleoli of intestinal epithelial cells via a carrier-dependent process.

Shiga toxin (Stx) produced by the invasive Shigella dysenteriae serotype 1 (S. dysenteriae1) causes gastrointestinal and kidney complications. It has been assumed that Stx is released intracellularly after enterocyte invasion by S. dysenteriae1. However, there is little information about Stx distribution inside S. dysenteriae1-infected enterocytes. Here, we use intestinal epithelial T84 cells to characterize the trafficking of Stx delivered into the cytosol, in ways that mimic aspects of S. dysenteriae1 infection. We find that cytoplasmic Stx is transported into nucleoli. Stx nucleolar movement is carrier- and energy-dependent. Stx binding to the nucleoli of normal human enterocytes in vitro supports possible roles for nucleolar trafficking in toxin-induced intestinal pathology.

NHE3 regulatory complexes.

The epithelial brush border Na/H exchanger NHE3 is active under basal conditions and functions as part of neutral NaCl absorption in the intestine and renal proximal tubule, where it accounts for the majority of total Na absorbed. NHE3 is highly regulated. Both stimulation and inhibition occur post-prandially. This digestion related regulation of NHE3 is mimicked by multiple extracellular agonists and intracellular second messengers. The regulation of NHE3 depends on its C-terminal cytoplasmic domain, which acts as a scaffold to bind multiple regulatory proteins and links NHE3 to the cytoskeleton. The cytoskeletal association occurs by both direct binding to ezrin and by indirect binding via ezrin binding to the C-terminus of the multi-PDZ domain containing proteins NHERF1 and NHERF2. This is a review of the domain structure of NHE3 and of the scaffolding function and role in the regulation of NHE3 of the NHE3 C-terminal domain.

Urine electrolyte, mineral, and protein excretion in NHERF-2 and NHERF-1 null mice.

The adaptor proteins sodium/hydrogen exchanger regulatory factor (NHERF)-1 and NHERF-2 have overlapping tissue distribution in renal cells and overlapping specificity in their binding to renal transporters and other proteins. To compare the kidney-specific differences in the function of these adaptor proteins, NHERF-1 and NHERF-2 null mice were compared with wild-type control mice. In NHERF-2 null mice, the renal proximal tubule abundance and distribution of NHERF-1 and NHERF-3 were not different from those in wild-type animals. The glomerular expression of podocalyxin and ZO-1 also did not differ. NHERF-1 null mice had increased urinary excretion of phosphate, calcium, and uric acid compared with wild-type control and NHERF-2 null mice. Because of the association between NHERF-2 and podocalyxin in glomeruli and ClC-5 in the renal proximal tubule, the urinary excretion of protein was determined. There were no differences in the urinary excretion of protein or low-molecular-weight proteins between wild-type control, NHERF-1(-/-), and NHERF-2(-/-) mice. These studies indicate that the increased urinary excretion of phosphate and uric acid are specific to NHERF-1 null mice and highlight the fact that predictions about the role of adaptor proteins such as the NHERF proteins obtained from studies of model cell systems must be confirmed in whole animals.

Tissue-specific regulation of sodium/proton exchanger isoform 3 activity in Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) null mice. cAMP inhibition is differentially dependent on NHERF1 and exchange protein directly activated by cAMP in ileum versus proximal tubule.

The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.

Na+/H+ exchanger NHE3 activity and trafficking are lipid Raft-dependent.

A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3.

The lateral mobility of NHE3 on the apical membrane of renal epithelial OK cells is limited by the PDZ domain proteins NHERF1/2, but is dependent on an intact actin cytoskeleton as determined by FRAP.

The epithelial brush border (BB) Na+/H+ exchanger, NHE3, plays a major role in transcellular Na+ absorption in the renal proximal tubule. NHE3 activity is rapidly regulated by neurohumoral substances and growth factors via changes in its amount on the BB by a process partially involving vesicle trafficking. The PDZ domain-containing proteins, NHERF1/2, are scaffold proteins that link NHE3 to the actin cytoskeleton via their binding to both ezrin and NHE3. NHERF1/2 interact with both an internal C-terminal domain of NHE3 and the N-terminus of ezrin. We used fluorescence recovery after photobleaching (FRAP) to study the effect of NHERF1/2 on NHE3 mobility in the brush border of opossum kidney (OK) proximal tubule cells. A confocal microscope was used to allow the selective study of apical membrane versus intracellular NHE3. A chimera of NHE3-EGFP was transiently expressed in OK cells and its lateral diffusion in the apical membrane was measured with FRAP and confocal microscopy at 37 degrees C. The contribution of intracellular NHE3-EGFP to recovery on the OK surface not directly over the juxtanuclear area (non-JN) was negligible as exposure to the water soluble crosslinker BS3 (10 mM) at 4 degrees C resulted in no recovery of this component of surface NHE3-EGFP after photobleaching. The mobile fraction (Mf) of apical NHE3-EGFP was 47.5+/-2.2%; the effective diffusion coefficient (Deff) was (2.2+/-0.3) x10(-10) cm2/second. Overexpression of NHERF2 in OK cells decreased the Mf to 29.1+/-3.1% without changing Deff. In the truncation mutant, NHE3585-EGFP (aa 1-585), which lacks the NHERF1/2 binding domain, Mf increased to 66.4+/-2.2%, with no change in Deff, whereas NHE3660-EGFP, which binds NHERF1/2, had Mf (48.3+/-3.0%) and Deff both similar to full-length NHE3. These results are consistent with the PDZ domain proteins NHERF1 and NHERF2 scaffolding NHE3 in macromolecular complexes in the apical membrane of OK cells under basal conditions, which limits the lateral mobility of NHE3. It is probable that this is one of the mechanisms by which NHERF1/2 affects rapid regulation of NHE3 by growth factors and neurohumoral mediators. By contrast, disrupting the actin cytoskeleton by latrunculin B treatment (0.05 microM, 30 minutes) reduced the NHE3 Mf (21.9+/-4.5%) without altering the Deff. Therefore the actin cytoskeleton, independently of NHERF1/2 binding, is necessary for apical membrane mobility of NHE3.