Icariside II, a Prenyl-Flavonol, Alleviates Inflammatory and Neuropathic Pain by Inhibiting T-Type Calcium Channels and USP5-Cav3.2 Interactions.

Cav3.2 channels play an important role in the afferent nociceptive pathway, which is responsible for both physiological and pathological pain transmission. Cav3.2 channels are upregulated during neuropathic pain or peripheral inflammation in part due to an increased association with the deubiquitinase USP5. In this study, we investigated nine naturally occurring flavonoid derivatives which we tested for their abilities to inhibit transiently expressed Cav3.2 channels and their interactions with USP5. Icariside II (ICA-II), one of the flavonols studied, inhibited the biochemical interactions between USP5 and Cav3.2 and concomitantly and effectively blocked Cav3.2 channels. Molecular docking analysis predicts that ICA-II binds to the cUBP domain and the Cav3.2 interaction region. In addition, ICA-II was predicted to interact with residues in close proximity to the Cav3.2 channel's fenestrations, thus accounting for the observed blocking activity. In mice with inflammatory and neuropathic pain, ICA-II inhibited both phases of the formalin-induced nocifensive responses and abolished thermal hyperalgesia induced by injection of complete Freund's adjuvant (CFA) into the hind paw. Furthermore, ICA-II produced significant and long-lasting thermal anti-hyperalgesia in female mice, whereas Cav3.2 null mice were resistant to the action of ICA-II. Altogether, our data show that ICA-II has analgesic activity via an action on Cav3.2 channels.

A Synthetically Accessible Small-Molecule Inhibitor of USP5-Cav3.2 Calcium Channel Interactions with Analgesic Properties.

Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund's adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesics─well suited to rapid structure-activity relationship studies─that target the Cav3.2/USP5 interface.

SUMOylation regulates USP5-Cav3.2 calcium channel interactions.

Cav3.2 calcium channels play a key role in nociceptive signaling in the primary afferent pain pathway. We have previously reported the regulation of Cav3.2 calcium channels by the deubiquitinase USP5 and its importance for regulating peripheral transmission of pain signals. Here we describe the regulation of the Cav3.2-USP5 interaction by SUMOylation. We show that endogenous USP5 protein expressed in dorsal root ganglia undergoes SUMOylation, and the level of USP5 SUMOylation is reduced following peripheral nerve injury. SUMO prediction software identified several putative lysines that have the propensity to be targets for SUMO conjugation. A series of single lysine substitutions in an mCherry tagged USP5 construct followed by expression in tsA-201 cells identified lysine K113 as a key target for USP5 SUMO2/3 modification. Finally, Cav3.2 calcium channel immunoprecipitates revealed a stronger interaction of Cav3.2 with a SUMO2/3 resistant USP5-K113R mutant, indicating that SUMO2/3 modification of USP5 reduces its affinity for the calcium channel Cav3.2. Collectively, our data suggest that dysregulation of USP5 SUMOylation after peripheral nerve injury may contribute to the well described alteration in Cav3.2 channel activity during neuropathic pain states.

Cav3.2 calcium channel interactions with the epithelial sodium channel ENaC.

This study describes the functional interaction between Cav3.2 calcium channels and the Epithelial Sodium Channel (ENaC). ß-ENaC subunits showed overlapping expression with endogenous Cav3.2 calcium channels in the thalamus and hypothalamus as detected by immunostaining. Moreover, ß- and γ-ENaC subunits could be co-immunoprecipitated with Cav3.2 calcium channels from brain lysates, dorsal horn and lumbar dorsal root ganglia. Mutation of a cluster of lysines present in the intracellular N-terminus region of ß-ENaC (K4R/ K5R/ K9R/ K16R/ K23R) reduced interactions with Cav3.2 calcium channels. Αßγ-ENaC channels enhanced Cav3.2 calcium channel trafficking to the plasma membrane in tsA-201 cells. This effect was reciprocal such that Cav3.2 channel expression also enhanced ß-ENaC trafficking to the cell surface. T-type current density was increased when fully assembled αßγ-ENaC channels were transiently expressed in CAD cells, a neuronal derived cell line. Altogether, these findings reveal ENaC as an interactor and potential regulator of Cav3.2 calcium channels expressed in neuronal tissues.

Selective inhibition of CaV3.2 channels reverses hyperexcitability of peripheral nociceptors and alleviates postsurgical pain.

Pain-sensing sensory neurons of the dorsal root ganglion (DRG) can become sensitized or hyperexcitable in response to surgically induced peripheral tissue injury. We investigated the potential role and molecular mechanisms of nociceptive ion channel dysregulation in acute pain conditions such as those resulting from skin and soft tissue incision. We used selective pharmacology, electrophysiology, and mouse genetics to link increased current densities arising from the CaV3.2 isoform of T-type calcium channels (T-channels) to nociceptive sensitization using a clinically relevant rodent model of skin and deep tissue incision. Furthermore, knockdown of the CaV3.2-targeting deubiquitinating enzyme USP5 or disruption of USP5 binding to CaV3.2 channels in peripheral nociceptors resulted in a robust antihyperalgesic effect in vivo and substantial T-current reduction in vitro. Our study provides mechanistic insight into the role of plasticity in CaV3.2 channel activity after surgical incision and identifies potential targets for perioperative pain that may greatly decrease the need for narcotics and potential for drug abuse.

T-type calcium channels functionally interact with spectrin (α/ß) and ankyrin B.

This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/ß) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/ß) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/ß) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.

Identification of interleukin-1 beta as a key mediator in the upregulation of Cav3.2-USP5 interactions in the pain pathway.

Abstract: We recently reported that nerve injury or peripheral inflammation triggers an upregulation of the deubiquitinase, USP5 in mouse dorsal root ganglion and spinal dorsal horn. This leads to dysregulated ubiquitination of Cav3.2 T-type calcium channels, thus increasing Cav3.2 channel plasma membrane expression and nociceptive signaling in the primary afferent pain pathway. This phenomenon could be recapitulated by noninvasive, optogenetic activation of transient receptor potential vanilloid-1­expressing nociceptors, indicating that neuronal activity is a key player in this process. Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels. Here, we report that gene expression, as well as protein levels for interleukin-1 beta and the endogenous interleukin-1 receptor-I antagonist, IL-1Ra are unaltered following conditioning stimulation of optogenetically targeted cutaneous nociceptors, indicating that neuronal activity is not a driver of interleukin-1 beta signaling. In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn. Moreover, disruption of the interaction between USP5 and Cav3.2 with TAT peptides suppressed acute nocifensive responses produced by interleukin-1 beta, which was similar to that achieved by elimination of T-type channel activity with the channel blockers, mibefradil, or TTA-A2. Finally, this upregulation could be maintained in dorsal root ganglion neuron cultures exposed overnight to interleukin-1 beta, while the copresence of interleukin-1 receptor antagonist or the dampening of neuronal cell activity with tetrodotoxin attenuated this response. Altogether, our findings identify interleukin-1 beta as an upstream trigger for the upregulation of interactions between USP5 and Cav3.2 channels in the pain pathway, presumably by triggering increased firing activity in afferent fibers.

TRPV1 Nociceptor Activity Initiates USP5/T-type Channel-Mediated Plasticity.

Peripheral nerve injury and tissue inflammation result in upregulation of the deubiquitinase USP5, thus causing a dysregulation of T-type calcium channel activity and increased pain sensitivity. Here, we have explored the role of afferent fiber activity in this process. Conditioning stimulation of optogenetically targeted cutaneous TRPV1 expressing nociceptors, but not that of non-nociceptive fibers, resulted in enhanced expression of USP5 in mouse dorsal root ganglia and spinal dorsal horn, along with decreased withdrawal thresholds for thermal and mechanical stimuli that abated after 24 hr. This sensitization was drastically reduced by an interfering peptide that prevented USP5-Cav3.2 association. Sensitization was relieved by pharmacological block of TRPV1 afferents, but not of myelinated neurons. In spinal cord slice recordings, we could optogenetically trigger an activity-dependent potentiation of presynaptic neurotransmission in the spinal dorsal horn that relied on Cav3.2 channel activity. This neuronal-activity-induced USP5 upregulation may underlie a protective, transient sensitization of the pain pathway.

A cell-permeant peptide corresponding to the cUBP domain of USP5 reverses inflammatory and neuropathic pain.

BACKGROUND: Cav3.2 T-type calcium currents in primary afferents are enhanced in various painful pathological conditions, whereas inhibiting Cav3.2 activity or expression offers a strategy for combating the development of pain hypersensitivity. We have shown that Cav3.2 channel surface density is strongly regulated by the ubiquitination machinery and we identified the deubiquitinase USP5 as a Cav3.2 channel interacting protein and regulator of its cell surface expression. We also reported that USP5 is upregulated in chronic pain conditions. Conversely, preventing its binding to the channel in vivo mediates analgesia in inflammatory and neuropathic pain models. RESULTS: To identify which USP5 domain is responsible for the interaction, we used a series of USP5-derived peptides corresponding to different regions in nUBP, cUBP, UBA1, and UBA2 domains to outcompete full length USP5. We identified a stretch of amino acid residues within the cUBP domain of USP5 as responsible for binding to Cav3.2 calcium channels. Based on this information, we generated a TAT-cUBP1-USP5 peptide that could disrupt the Cav3.2/USP5 interaction in vitro and tested its physiological effect in well-established models of persistent inflammatory pain (CFA test) and chronic mononeuropathy and polyneuropathy in mice (partial sciatic nerve injury and the (ob/ob) diabetic spontaneous neuropathic mice). Our results reveal that the TAT-cUBP1-USP5 peptide attenuated mechanical hyperalgesia induced by both Complete Freund's Adjuvant and partial sciatic nerve injury, and thermal hyperalgesia in diabetic neuropathic animals. In contrast, Cav3.2 null mice were not affected by the peptide in the partial sciatic nerve injury model. Cav3.2 calcium channel levels in diabetic mice were reduced following the administration of the TAT-cUBP1-USP5 peptide. CONCLUSIONS: Our findings reveal a crucial region in the cUBP domain of USP5 that is important for substrate recognition and binding to the III-IV linker of Cav3.2 channels. Targeting the interaction of this region with the Cav3.2 channel can be exploited as the basis for therapeutic intervention into inflammatory and neuropathic pain.

The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity.

T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.

Energetic and structural basis for activation of the epithelial sodium channel by matriptase.

Limited proteolysis, accomplished by endopeptidases, is a ubiquitous phenomenon underlying the regulation and activation of many enzymes, receptors, and other proteins synthesized as inactive precursors. Serine proteases make up one of the largest and most conserved families of endopeptidases involved in diverse cellular activities, including wound healing, blood coagulation, and immune responses. Heteromeric α,ß,γ-epithelial sodium channels (ENaC) associated with diseases like cystic fibrosis and Liddle's syndrome are irreversibly stimulated by membrane-anchored proteases (MAPs) and furin-like convertases. Matriptase/channel activating protease-3 (CAP3) is one of the several MAPs that potently activate ENaC. Despite identification of protease cleavage sites, the basis for the enhanced susceptibility of α- and γ-ENaC to proteases remains elusive. Here, we elucidate the energetic and structural bases for activation of ENaC by CAP3. We find a region near the γ-ENaC furin site that has previously not been identified as a critical cleavage site for CAP3-mediated stimulation. We also report that CAP3 mediates cleavage of ENaC at basic residues downstream of the furin site. Our results indicate that surface proteases alone are sufficient to fully activate uncleaved ENaC and explain how ENaC in epithelia expressing surface-active proteases can appear refractory to soluble proteases. Our results support a model in which proteases prime ENaC for activation by cleaving at the furin site, and cleavage at downstream sites is accomplished by membrane surface proteases or extracellular soluble proteases. On the basis of our results, we propose a dynamics-driven "anglerfish" mechanism that explains less stringent sequence requirements for substrate recognition and cleavage by matriptase than by furin.

Activation of the epithelial sodium channel by the metalloprotease meprin ß subunit.

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin ß (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin ß or α/ß in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin ß, but not the α subunit. Meprin ß promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin ß regulates the activity of ENaC in a metalloprotease-dependent fashion.

The Cavß subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels.

It is well established that the auxiliary Cavß subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavß subunit increased membrane expression of Cav1.2 channels by preventing the entry of the channels into the endoplasmic reticulum-associated protein degradation (ERAD) complex. Without Cavß, Cav1.2 channels underwent robust ubiquitination by the RFP2 ubiquitin ligase and interacted with the ERAD complex proteins derlin-1 and p97, culminating in targeting of the channels to the proteasome for degradation. On treatment with the proteasomal inhibitor MG132, Cavß-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavß interfered with ubiquitination and targeting of the channel to the ERAD complex, thereby facilitating export from the endoplasmic reticulum and promoting expression on the cell surface. Thus, Cavßß regulates the ubiquitination and stability of the calcium channel complex.

The cystic fibrosis transmembrane conductance regulator impedes proteolytic stimulation of the epithelial Na+ channel.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl(-) secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na(+) absorption. The mechanistic link between missing CFTR and increased Na(+) absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na(+) channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na(+) absorption.

SPLUNC1 expression reduces surface levels of the epithelial sodium channel (ENaC) in Xenopus laevis oocytes.

Throughout the body, the epithelial Na(+) channel (ENaC) plays a critical role in salt and liquid homeostasis. In cystic fibrosis airways, for instance, improper regulation of ENaC results in hyperabsorption of sodium that causes dehydration of airway surface liquid. This dysregulation then contributes to mucus stasis and chronic lung infections. ENaC is known to undergo proteolytic cleavage, which is required for its ability to conduct Na(+) ions. We have previously shown that the short, palate lung and nasal epithelial clone (SPLUNC1) binds to and inhibits ENaC in both airway epithelia and in Xenopus laevis oocytes. In this study, we found that SPLUNC1 was more potent at inhibiting ENaC than either SPLUNC2 or long PLUNC1 (LPLUNC1), two other PLUNC family proteins that are also expressed in airway epithelia. Furthermore, we were able to shed light on the potential mechanism of SPLUNC1's inhibition of ENaC. While SPLUNC1 did not inhibit proteolytic activity of trypsin, it significantly reduced ENaC currents by reducing the number of ENaCs in the plasma membrane. A better understanding of ENaC's regulation by endogenous inhibitors may aid in the development of novel therapies designed to inhibit hyperactive ENaC in cystic fibrosis epithelia.

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage.

Many epithelia, including the superficial epithelia of the airways, are thought to secrete "volume sensors," which regulate the volume of the mucosal lining fluid. The epithelial Na(+) channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na(+) and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.

ENaC proteolytic regulation by channel-activating protease 2.

Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na(+) absorption across epithelial surfaces throughout the body. Excessive Na(+) absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na(+) absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (alpha-K561, beta-R503, and gamma-R515). Sites in alpha-ENaC at K149/R164/K169/R177 and furin-consensus sites in alpha-ENaC (R205/R231) and gamma-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in gamma-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (alpha,beta,gammaR138A/Q) completely abolished both the Na(+) current (I(Na)) and a 75-kD gamma-ENaC fragment at the cell surface stimulated by CAP2. Replacement of gamma-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced I(Na) and the 75-kD gamma-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in gamma-ENaC.

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis.

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.

Agonist-induced interactions between angiotensin AT1 and epidermal growth factor receptors.

In rat hepatic C9 cells, angiotensin II (Ang II)-induced activation of angiotensin type 1 (AT(1)) receptors (AT(1)-Rs) stimulates extracellular signal-regulated kinase (ERK) 1/2 phosphorylation via transactivation of the endogenous epidermal growth factor (EGF) receptor (EGF-R) by a protein kinase C (PKC) delta/Src/Pyk2-dependent pathway. This leads to phosphorylation of the EGF-R as well as its subsequent internalization. On the other hand, EGF-induced activation of the EGF-R in C9 cells was found to cause phosphorylation of the AT(1)-R. This was prevented by selective inhibition of the intrinsic tyrosine kinase activity of the EGF-R by AG1478 [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline] and was reduced by inhibition of PKC and phosphoinositide 3-kinase. EGF-induced AT(1)-R phosphorylation was associated with a decrease in membrane-associated AT(1)-Rs and a reduced inositol phosphate response to Ang II. Agonist activation of endogenous AT(1)-Rs and EGF-Rs induced the formation of a multireceptor complex containing both the AT(1)-R and the transactivated EGF-R. The dependence of these responses on caveolin was indicated by the finding that cholesterol depletion of C9 cells abolished Ang II-induced inositol phosphate production, activation of Akt/PKB and ERK1/2, and AT(1)-R internalization. Confocal microscopy demonstrated that caveolin-1 was endogenously phosphorylated and was distributed on the plasma membrane in patches that undergo redistribution during Ang II stimulation. Agonist-induced phosphorylation and association of caveolin 1 with the AT(1)-R was observed, consistent with a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. The EGF-induced AT(1)-R/caveolin association was abolished by AG1478, suggesting that activation of the EGF-R promotes the association of caveolin and the AT(1)-R.