AT1R Internalization Research Articles

ATRAP, a receptor-interacting modulator of kidney physiology, as a novel player in blood pressure and beyond.

Pathological activation of kidney angiotensin II (Ang II) type 1 receptor (AT1R) signaling stimulates tubular sodium transporters, including epithelial sodium channels, to increase sodium reabsorption and blood pressure. During a search for a means to functionally and selectively modulate AT1R signaling, a molecule directly interacting with the carboxyl-terminal cytoplasmic domain of AT1R was identified and named AT1R-associated protein (ATRAP/Agtrap). We showed that ATRAP promotes constitutive AT1R internalization to inhibit pathological AT1R activation in response to certain stimuli. In the kidney, ATRAP is abundantly distributed in epithelial cells along the proximal and distal tubules. Results from genetically engineered mice with modified ATRAP expression show that ATRAP plays a key role in the regulation of renal sodium handling and the modulation of blood pressure in response to pathological stimuli and further suggest that the function of kidney tubule ATRAP may be different between distal tubules and proximal tubules, implying that ATRAP is a target of interest in hypertension.

A Limited or a Transient AT1R Internalization Induced by Two Discriminating Agonists Result in Distinct Downstream AT1R Signaling: ERK1/2 and Akt/PKB

Background Agonist-induced AT1R Internalization, results from sequestration of the agonist-receptor complex into “clathrin vesicles”, depending on the internalization proteins: β-arrestin, clathrin. In this regard, we have shown that a 15,000 kDa polymer of Ang II (Ang II-POL) at maximal concentration, upon a sustained action on the AT1R of rat coronary endothelial luminal membrane (CELM) produced: a sustained inotropic effect, a lack of endothelial AT1R internalization, enhanced membranal recruitment of β-arrestin1/2, without clathrin recruitment a lack of clathrin-vesicles formation. In contrast, the monomer Ang II (1 kDa) inotropic effects shows desensitization, paralleled by endothelial AT1R internalization, and the well-known gradual membranal recruitment of the internalization proteins β-arrestin1/2 and clathrin. These results clearly show that Ang II and Ang II-POL distinctly activate AT1R as reflected by their different recruitment ways of the internalization proteins, which likely result in distinct downstream AT1R signaling: ERK1/2 and Akt/PKB phosphorylation. We studied this canonical signaling pathways triggered by AT1R activation, with both agonists. Hypothesis The distinct rates of membrane-recruitment of the internalization proteins; β-arrestin and clathrin by Ang II and Ang II-POL likely results also in distinct downstream AT1R signaling: ERK1/2, Akt/PKB phosphorylation. Hypothesis test C9 cells were used as a model of cytoplasmic events. Cells were stimulated either with maximal concentrations of Ang II or Ang II-POL and under various conditions were determined the changes in ERK1/2 and Akt/PKB phosphorylation: 1) at various times, 2) in the presence of effect of AT1R and AT2R antagonist and 3) in the presence of various PI3K and MEK1 inhibitors. Results 1) Ang II as well as Ang II-POL caused, via an AT1R activation, a time dependent phosphorylation of ERK1/2 and Akt/PKB. These time-dependent responses, 2) in the case of Akt the response by Ang II was transient while for Ang II-POL rose to a plateau and sustained, but, 3) in the case of ERK1/2’s for both agonists rose followed by a gradual drop and were equally transient. In addition, 4) PI3K inhibitors blocked the Akt responses induced Ang II and Ang II-POL, however, in the case of ERK1/2, the inhibitors only blocked the effects of Ang II-POL but not Ang II's. Conclusion The distinct rates of membrane-recruitment of the internalization proteins; β-arrestin and clathrin by Ang II and Ang II-POL are associated with distinct downstream AT1R signaling: ERK1/2, Akt/PKB. For example, a sustained AT1R activation or recruitments, enhanced for β-arrestin1/2 and inhibited for clathrin prevent desensitization of Akt/PKB; in addition, PI3K dependent phosphorylation of ERK. Clearly, AngII-POL constitutes a powerful tool for understanding the internalization and intracellular signaling mechanisms, induced by sustained AT1R activation.

A Limited Vs a Transient AT1R Internalization Indicates that Distinct Mechanisms of AT1R Activation are Possible

A Limited Vs a Transient AT1R Internalization Indicates that Distinct Mechanisms of AT1R Activation are Possible

RECEPTOR INTERACTING MOLECULE ATRAP AS MODULATOR OF RENAL SODIUM HANDLING AND PATHOLOGICAL BLOOD PRESSURE RESPONSE

Objective: Pathological activation of renal angiotensin II (Ang II) type 1 receptor (AT1R) signaling exerts stimulates renal tubular Na+ transporters including epithelial Na+ channel to increase renal sodium reabsorption. Design and method: In the course of an investigational search for a means to functionally and selectively modulate AT1R signaling for that purpose, a molecule directly interacting with the carboxyl-terminal cytoplasmic domain of AT1R was identified by employing yeast two-hybrid screening of a mouse kidney cDNA library and named AT1R-associated protein (ATRAP). Results: We showed that ATRAP promotes constitutive AT1R internalization so as to inhibit pathological AT1R activation in response to certain stimuli. In the kidney, ATRAP is abundantly distributed in epithelial cells along the renal tubules. The results employing genetic engineered mice with modified ATRAP expression showed that ATRAP plays a key role in the regulation of renal sodium handling and the modulation of blood pressure in response to pathological stimuli such as chronic Ang II infusion dietary high salt loading and 5/6 nephrectomy. Conclusions: Accumulating findings concerning functional and pathological roles of the novel receptor-interacting molecule ATRAP suggest that ATRAP would be a target of interest for hypertension in the cardiovascular and renal continuum.

Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury.

Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, β-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis. We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII. We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR. Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Limited AT1 Receptor Internalization Is a Novel Mechanism Underlying Sustained Vasoconstriction Induced by AT1 Receptor Autoantibody From Preeclampsia.

BackgroundAngiotensin II type 1 receptor (AT 1R) autoantibody (AT1‐AA) was first identified as a causative factor in preeclampsia. Unlike physiological ligand angiotensin II (Ang II), AT1‐AA can induce vasoconstriction in a sustained manner, causing a series of adverse effects, such as vascular injury and poor placental perfusion. However, its underlying mechanisms remain unclear. Here, from the perspective of AT 1R internalization, the present study investigated the molecular mechanism of sustained vasoconstriction induced by AT 1R autoantibody.Methods and ResultsIn the current study, we used the vascular‐ring technique to determine that AT1‐AA‐positive IgG, which was obtained from the sera of preeclamptic patients, induced long‐term vasoconstriction in endothelium‐intact or endothelium‐denuded rat thoracic arteries. The effect was caused by prolonged activation of AT 1R downstream signals in vascular smooth muscle cells, including Ca2+, protein kinase C, and extracellular signal‐regulated kinase 1 and 2. Then, using subcellular protein fractionation, cell surface protein biotinylation, and total internal reflection fluorescence, we found that AT1‐AA‐positive IgG resulted in significantly less AT 1R internalization than in the Ang II treatment group. Moreover, through use of fluorescent tracing and bioluminescence resonance energy transfer, we found that AT1‐AA‐positive IgG cannot induce the recruitment of β‐arrestin1/2, which mediated receptor internalization. Then, the effect of sustained AT 1R activation induced by AT1‐AA‐positive IgG was rescued by overexpression of β‐arrestin2.ConclusionsThese data suggested that limited AT 1R internalization resulting from the inhibition of β‐arrestin1/2 recruitment played an important role in sustained vasoconstriction induced by AT1‐AA‐positive IgG, which may set the stage for avoiding AT 1R overactivation in the management of preeclampsia.

An angiotensin II type 1 receptor binding molecule has a critical role in hypertension in a chronic kidney disease model

Angiotensin II type 1 receptor-associated protein (ATRAP) promotes AT1R internalization along with suppression of hyperactivation of tissue AT1R signaling. Here, we provide evidence that renal ATRAP plays a critical role in suppressing hypertension in a mouse remnant kidney model of chronic kidney disease. The effect of 5/6 nephrectomy on endogenous ATRAP expression was examined in the kidney of C57BL/6 and 129/Sv mice. While 129/Sv mice with a remnant kidney showed decreased renal ATRAP expression and developed hypertension, C57BL/6 mice exhibited increased renal ATRAP expression and resistance to progressive hypertension. Consequently, we hypothesized that downregulation of renal ATRAP expression is involved in pathogenesis of hypertension in the remnant kidney model of chronic kidney disease. Interestingly, 5/6 nephrectomy in ATRAP-knockout mice on the hypertension-resistant C57BL/6 background caused hypertension with increased plasma volume. Moreover, in knockout compared to wild-type C57BL/6 mice after 5/6 nephrectomy, renal expression of the epithelial sodium channel α-subunit and tumor necrosis factor-α was significantly enhanced, concomitant with increased plasma membrane angiotensin II type 1 receptor in the kidneys. Thus, renal ATRAP downregulation is involved in the onset and progression ofblood pressure elevation caused by renal mass reduction,and implicates ATRAP as a therapeutic target forhypertension in chronic kidney disease.

CaV1.2 Interaction with AT1R Reduces Receptor Internalization

The cardiac L type calcium channel is a multi-subunit complex that includes CaV1.2 as the pore-forming subunit that co-assembles with the auxiliary CaVa2d1 subunit and with the CaVb subunit. The main role of these auxiliary subunits is to modify the amount of channels inserted in the plasma membrane. Similarly, trafficking of these channels is controlled by direct interaction with other proteins such as some G-protein coupled receptors (GPCR). Angiotensin II (AngII) type I receptor (AT1R) is a GPCR well known to be involved in the intracellular calcium regulation at the heart, modulating cardiomyocytes contractibility. In this work we show, by co-immunoprecipitation, that AT1R forms a macro-complex with CaV1.2 in primary culture of newborn rat cardiomyocytes and in an heterologous system. Standard biochemical approaches were used to demonstrate that these proteins colocalize at the plasma membrane and to determine the region of CaV1.2 responsible for this interaction. The functional impact of this complex was studied in terms of L-type currents inhibition and in terms of internalization of AT1R. Although inhibition of endogenous L-type current in cardiomyocytes was found to be dependent on the CaVb2 transcriptional start site (TSS) expressed, the presence of the macro-complex was found to be independent on the variant expressed. The effect of this interaction over AT1R internalization was studied in cells stimulated with AngII at different times. As expected, in cell expressing only the AT1R and stimulated with AngII, the receptor is readily internalized after 15 min, however, in cells expressing also CaV1.2, AT1R internalization (and CaV1.2) is observed only after 30 minutes of AngII stimulation. Overall, our results demonstrate that AT1R interaction with CaV1.2 increases the residence time at the plasma membrane of the receptor and induce L-type channel internalization after prolonged AngII exposure.

Angiotensin II Type 1 Receptor Binding Molecule ATRAP as a Possible Modulator of Renal Sodium Handling and Blood Pressure in Pathophysiology.

Exaggerated activation of the renin-angiotensin system via tissue angiotensin II (Ang II) type 1 receptor (AT1R) signaling exerts detrimental effects on cardiovascular, renal and endocrine systems to provoke hypertension and related target organ damage. On the other hand, accumulated research evidence of both basic and clinical studies shows that physiological AT1R signaling also plays an indispensable role for the normal organ development such as the kidney and the maintenance of cardiovascular and renal homeostasis. Such functional diversity of AT1R signaling prompts us to seek a new strategy of selective modulation of AT1R signaling in pathophysiology. In the course of an investigational search for a means to functionally and selectively modulate AT1R signaling for that purpose, a molecule directly interacting with the carboxyl-terminal cytoplasmic domain of AT1R was identified by employing yeast two-hybrid screening of a mouse kidney cDNA library and named AT1R-associated protein (ATRAP). The results of functional analysis showed that ATRAP promotes constitutive AT1R internalization in cultured cells and inhibits Ang II-mediated pathological response in mouse distal convoluted cells. The ATRAP is expressed in a variety of tissues including the kidney where ATRAP is abundantly distributed in epithelial cells along the renal tubules. The results employing genetic engineered mice with modified ATRAP expression showed that ATRAP plays a key role in the regulation of renal sodium handling and the modulation of blood pressure in response to pathological stimuli such as chronic Ang II infusion, and suggest ATRAP to be a target of interest.

Abstract 022: A Short Open Reading Frame in the Angiotensin Type 1a Receptor 5' Leader Sequence Increases the Rate of Receptor Internalization

Background: We recently found that a seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5’ leader sequence (5'LS) of the angiotensin type 1a receptor (AT1aR) mRNA inhibits AT1aR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) without having any effect on the AT1aR-inositol trisphosphate protein kinase C pathway. To investigate the mechanism by which PEP7 selectively inhibits the AT1aR-Erk1/2 signaling cascade, the start codon of the sORF was mutated at adenine -108 (A-108 to T-108) to create E1,2(-108T),3-AT1aR followed by cloning the E1,2,3-AT1aR and the mutant E1,2(-108T),3-AT1aR into the pEGFP-N2 plasmid. Methods: Human embryonic kidney 293 (HEK-293) cells were transfected with E1,2,3-AT1aR (intact sORF construct) or E1,2(-108T),3-AT1aR (disrupted sORF construct) by lipofectamine. Forty eight to seventy two hours later, live cell time lapse images were collected before and after treatment with Ang II (100 nM) using a TE300 Spinning disk laser scanning confocal microscope. Image analysis was performed using Velocity software. Results: Within 5 min of Ang II stimulation, punctae formed and moved throughout the cell membrane in cells transfected with both EGFP tagged receptors; however, even though the punctae represent the localized accumulation of identical AT1aR proteins, there were distinct differences in the intensity and time course of punctae. The rate of vesicle formation after Ang II treatment was markedly decreased by disrupting the PEP7 sORF [t1/2 (s): E1,2,3-AT1aR, 203s (N=21) vs E1,2(-108T),3-AT1aR, 328s (N=14); p<0.0001]. Conclusion: The PEP7 sORF facilitates Ang II-induced AT1R internalization. These findings suggest that we have uncovered a new mechanism governing agonist-induced AT1aR cellular trafficking that could have implications not only for regulation of AT1aR signaling cascades but also for other trafficking proteins that contain an upstream sORF within their 5'LS.

CORONARY VASOCONSTRICTION INDUCED BY POLYMERS OF ANG II IS PROPORTIONAL TO LUMINAL ENDOTHELIAL AT1 RECEPTORS DENSITY

We have shown that intravascular AngII does not diffuse across the coronary vessel wall and acts solely in coronary endothelial luminal membrane AngII receptors (CELM‐AT1R) and its sustained intracoronary infusion causes transient vasoconstriction effects (+V). This contrasted with the sustained +V by a large size, non‐diffusible polymer of AngII (15,000 kDa, AngIIPol) that unquestionably activate solely CELM‐AT1R. We also show that AngII transient +V paralleled a decrease of CELM‐AT1R density (AT1RD), but, for AngIIPol the AT1RD remained constant. HYPOTHESIS; transiently of +V effects depends directly on AT1RD both are inversely determined by the molecular weight (MW) of AT1R agonist. To test this hypothesis we synthesized AngIIPol's (included AngII) of various MW (kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15000. In isolated constant flow (8ml/min) perfused Langendorff guinea pig hearts AII‐Pol's were separately intracoronary given for 16 min and coronary perfusion pressure measure as an index V. All AngIIPol's caused the same maximal +V (+Vmax) that decayed with time at a rate and to a stable level of +V, both inversely proportional to MW. During AngIIpol's infusion AT1RD decayed parallel to +V; the relationship +V‐‐AT1RD was linear. Further dependence of +V on AT1RD was demonstrated by two successive administrations of AngIIPol's, the primary dose caused a +Vmax but the +Vmax to the secondary dose was depressed; a tachyphylaxis which was inversely proportional to the logarithm of MW. These results support our hypothesis and indicate that AT1R internalization is dependent on MW.

Molecular weight of different angiotensin II polymers directly determines: Density of endothelial membrane AT1 receptors and coronary vasoconstriction

We have shown that angiotensin II (Ang II) does not diffuse across the vessel wall, remaining intravascularly confined and acting solely on the coronary endothelial luminal membrane (CELM) receptors. A sustained intracoronary infusion of Ang II causes transient coronary vasoconstriction (desensitization) due to membrane internalization of CELM Ang II type 1 receptors (CELM-AT1R). In contrast, sustained intracoronary infusion of a non-diffusible polymer of Ang II (Ang II-Pol, 15,000kDa) causes a sustained vasoconstriction by preventing CELM-AT1R internalization. In addition, a sustained intracoronary infusion of Ang II leads to a depressed response following a secondary Ang II administration (tachyphylaxis) that is reversed by Ang II-Pol. These findings led us to hypothesize that the rate of desensitization, tachyphylaxis, and AT1R internalization were dependent on Ang II-Pol molecular weight. To test this hypothesis, we synthesized Ang II-Pols of the following molecular weights (in kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15,000. Vasoconstriction was measured following intracoronary infusion of Ang II-Pols in Langendorff-perfused guinea pig hearts at constant flow. The CELM protein fraction was extracted using the silica pellicle technique at different time points in order to determine the rate of AT1R internalization following each Ang II-Pol infusion. CELM-AT1R density was quantified by Western blot. We found that the rate of desensitization and the tachyphylaxis effect varied inversely with the molecular weight of the Ang II-Pols. Inversely proportional to the molecular weight of Ang II-Pol the CELM-AT1R density decreases over time. These results indicate that the mechanism responsible for the decreased rate of desensitization and tachyphylaxis by higher molecular weight Ang II polymers is due to reduction in the rate of CELM-AT1R internalization. These Ang II polymers would be valuable tools for studying the relationship between AT1R internalization and physiological effects.

Short-term memory in the heart: A road map for channel trafficking required

Short-term memory in the heart: A road map for channel trafficking required

Caveolae by interfering internalization of AT1 receptors regulate constrictions of isolated arterioles to Ang II

We hypothesized that caveolae regulate Ang II ‐ induced arteriolar constrictions by modulating the internalization of type 1 angiotensin II (Ang II) receptors (AT1R). Thus to assess the functional availability of AT1R, Ang II (10‐8 M) were administered in a repeated fashion (first and second applications) and the diameter of skeletal muscle arterioles (≈150 μm at 80 mmHg) were measured with videomicroscopy. Compared to the first application, Ang II‐induced constrictions were significantly reduced upon the second application (% max. constrictions: 59±3 vs. 37±2, P<0.05). Incubation of arterioles with concanavalin A (0.1 mM for 2 hours), known to inhibit AT1R internalization, had no effect on Ang II‐induced constrictions upon the first application (65±7%), but further significantly diminished constrictions to the second application (9±5%). Incubation of vessels with methyl β‐cyclodextrin (1 mM for 2 hours), known to disrupt caveolae, resulted in maintained Ang II‐induced arteriolar constrictions upon repeated applications (61±3 vs. 69±7, N.S.). We propose that internalization of AT1R is essential to restore active receptors on the surface of vascular smooth muscle cell, making them available for further stimulation and that caveolae by interfering with internalization limit AT1R availability and thus constrictions. (OTKA T48376, K71591, F48837; ETT 364/2006, and AHA 0855910D, 0735540T, USA)

ACTIVATION AND INTERNALIZATION OF CORONARY ENDOTHELIAL LUMINAL MEMBRANE (CELM) AT1 RECEPTOR CAN BE DISSOCIATED

Intracoronary prolonged infusion of Ang II and Ang II‐POL; a non diffusible large agonist cause a AT1R‐mediated positive inotropic effects with different time courses; Ang II‐POL effects are sustained and reversible, while Ang II's are transient indicating desensitization. In addition, Ang II induce tachyphylaxis to Ang II and Ang II‐POL, but Ang II‐POL does not. In vitro, desensitization and tachyphylaxis are associated with AT1R internalization. Thus, transient effects of sustained intracoronary Ang II result from gradual internalization of CELM's AT1R, not being the case for Ang II‐POL. To test this hypothesis we measured AT1R levels on CELM. CELM proteins were isolated from hearts perfused with Ang II for 0 to 10 min and from hearts perfused with ang II‐POL 0 to 15 min. AT1R band was identified by Western blot and its mass measured as band optical density (BOD) as respect to 0 time (BOD control = 1.0). Ang II perfusion caused a time‐dependent CELM's AT1R decrease that reached a value of 0.5 at 6 min which rose to reach 1.0 at 10 min. In contrast, Ang II‐POL CELM's AT1R values at various times remained as 1.0, i.e. there was not internalization. These results indicates that: Ang II activates CELM's AT1R and induces its internalization; while Ang II‐POL activates CELM's AT1R without inducing internalization

Src-dependent phosphorylation of β2-adaptin dissociates the β-arrestin–AP-2 complex

Beta-arrestins are known to act as endocytic adaptors by recruiting the clathrin adaptor protein 2 (AP-2) complex to G-protein-coupled receptors (GPCRs), linking them to clathrin-coated pits (CCPs) for internalization. They also act as signaling molecules connecting GPCRs to different downstream effectors. We have previously shown that stimulation of the angiotensin II (Ang II) type 1 receptor (AGTR1, hereafter referred to as AT1R), a member of the GPCR family, promotes the formation of a complex between beta-arrestin, the kinase Src and AP-2. Here, we report that formation of such a complex is involved in the AT1R-mediated tyrosine phosphorylation of beta2-adaptin, the subunit of AP-2 involved in binding beta-arrestin. We identify a crucial tyrosine residue in the ear domain of beta2-adaptin and show in vitro that the phosphorylation of this site regulates the interaction between beta-arrestin and beta2-adaptin. Using fluorescently tagged proteins combined with resonance energy transfer and image cross-correlation spectroscopy approaches, we show in live cells that beta2-adaptin phosphorylation is an important regulatory process for the dissociation of beta-arrestin-AP-2 complexes in CCPs. Finally, we show that beta2-adaptin phosphorylation is involved in the early steps of receptor internalization. Our findings not only unveil beta2-adaptin as a new Src target during AT1R internalization, but also support the role of receptor-mediated signaling in the control of clathrin-dependent endocytosis of receptors.

Posttranscriptional mechanisms contribute to osmotic regulation of ANG type 1 receptors in cultured rat renomedullary interstitial cells

Previously, we showed that ANG II receptors in cultured rat renomedullary interstitial cells (RMICs) are osmotically regulated (19). The current study examined the mechanisms underlying this osmotic regulation in RMICs cultured in isoosmotic (300 mosmol/kgH2O) and hyperosmotic (600 mosmol/kgH2O) conditions. Radioligand competition analysis coupled with RNase protection assays (RPA) and ligand-mediated receptor internalization studies revealed that RMICs primarily express the type 1a angiotensin receptor (AT(1a)R). When cultured under hyperosmotic conditions, the density (B(max)) of AT1R in RMIC membranes decreased by 31% [B(max) (pmol/mg protein): 300 mosmol/kgH2O, 6.44 +/- 0.46 vs. 600 mosmol/kgH2O, 4.42 +/- 0.37, n = 8, P < 0.01], under conditions in which no detectable changes in AT(1a)R mRNA expression or in the kinetics of ligand-mediated AT1R internalization were observed. RNA electromobility shift assays showed that RNA protein complex (RPC) formation between RMIC cytosolic RNA binding proteins and the 5' leader sequence (5'LS) of the AT(1a)R was increased 1.5-fold under hyperosmotic conditions [5'LS RPC (arbitrary units): 300 mosmol/kgH2O, 0.79 +/- 0.08 vs. 600 mosmol/kgH2O, 1.17 +/- 0.07, n = 4, P < 0.01]. These results suggest that the downregulation of AT(1a)R expression in RMICs cultured under hyperosmotic conditions is regulated at the posttranscriptional level by RNA binding proteins that interact within the 5'LS of the AT(1a)R mRNA. The downregulation of AT(1a)R expression under hyperosmotic conditions may be an important mechanism by which the activity of ANG II is regulated in the hyperosmotic renal medulla.

Tyrosine Kinase Inhibition Affects Type 1 Angiotensin II Receptor Intern Alization

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth factor receptor internalization. The type 1 angiotensin II receptor (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCI4 cells expressing rabbit AT1R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1–100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 #M], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 #M] or tyrphostin B-48 (TB-48) [50 #M], were assayed for acid-resistant specific 125I-Ang II binding, a measure of Ang II internalization. Both Gen (n=7) and TB-48 (n=3) inhibited AP 125I-Ang II internalization (80 ± 7% inhibition; p < 0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP 125I-Ang II endocytosis (n=3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n=5) or herbimycin A [150 ng/ml] (n=4) also inhibited MSC 125I-Ang II internalization (82 ± 11% inhibition; p < 0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.

Apical ANG II-stimulated PLA2 activity and Na+ flux: a potential role for Ca2+-independent PLA2.

Type 1 angiotensin II (ANG II) receptors (AT1R), which mediate proximal tubule (PT) salt and water reabsorption, undergo endocytosis and recycling. Prior studies in a PT-like model (LLC-PKcl4 cells expressing rabbit AT1R) (LLC-PK-AT1R cells) determined that quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor, and the haloenol lactone suicide substrate (HELSS), a Ca2+-independent PLA2 inhibitor, attenuated apical (AP) AT1R recycling. Further studies were undertaken to examine the association between AT1R endocytotic movement and PLA2 activity in this model. AP ANG II (100 nM) increased [3H]arachidonic acid ([3H]AA) release 4.4 +/- 0.38-fold in LLC-PK-AT1R cells cultured on permeable supports. Basolateral (BL) ANG II had no significant effect. Reversed-phase high-performance liquid chromatography confirmed that AP ANG II stimulated free [3H]AA release. Quinacrine, HELSS, and palmitoyl trifluoromethyl ketone, another Ca2+-independent PLA2 inhibitor, inhibited AP ANG II-stimulated [3H]AA release, as did inhibiting AP AT1R internalization with phenylarsine oxide. The role of HELSS-inhibitable AA release in ANG II-mediated 22Na flux was examined, given the effects of AT1R-mediated PLA2 activity on salt and water reabsorption. AP ANG II (100 nM) stimulated 22Na flux (AP--> BL), a response inhibited by HELSS. Thus, in this model, AP AT1R activated PLA2 with concomitant 22Na flux (AP --> BL), suggesting a link between AP AT1R endocytotic movement, AT1R-stimulated PLA2 activity, and 22Na flux in this model. The effects of HELSS suggest that Ca2+-independent PLA2 activity may be involved in this AP ANG II response.

Polarized rabbit type 1 angiotensin II receptors manifest differential rates of endocytosis and recycling.

Receptor-mediated endocytosis and recycling have been described for extrarenal angiotensin II (ANG II) receptors. In proximal tubule (PT) epithelia expressing polarized ANG II receptors, these processes have not been examined as thoroughly. We utilized a PT cell model, LLC-PKCl4 cells stably transfected with rabbit type 1 ANG II receptor (AT1R) cDNA, to investigate these properties. LLC-PK-AT1R cells expressed the rabbit AT1R transcript and displayed losartan-inhibitable specific 125I-labeled ANG II binding at apical (AP) and basolateral (BL) membranes when grown on permeable supports. AP AT1R internalized 125I-ANG II more rapidly than BL AT1R, and phenylarsine oxide treatment inhibited AP AT1R internalization without significantly affecting BL AT1R endocytosis. Pertussis toxin had no effect on AP or BL AT1R endocytosis. In addition, AP AT1R recovered specific 125I-ANG II binding after ANG II treatment (a measure of recycling). BL AT1R displayed minimal recovery of 125I-ANG II binding after ANG II pretreatment. These data suggested that AP AT1R enter endocytic/endosomal pathways. Phospholipase A2 (PLA2) activity has been linked to endosomal fusion in other systems, and PT brush-border membrane AT1R also have been associated with PLA2 activity. LLC-PK-AT1R cells were therefore treated with quinacrine, a nonspecific PLA2 inhibitor, or Compound I (CI), a selective Ca(2+)-independent PLA2 inhibitor, to determine if PLA2 activity was linked to AT1R recycling. Both quinacrine and CI decreased AP AT1R recycling without affecting BL AT1R recycling. Polarized AT1R in LLC-PKCl4 cells thus display differential rates of endocytosis and recycling.(ABSTRACT TRUNCATED AT 250 WORDS)