Thomas J. Thekkumkara

Angiotensin II Receptor Endocytosis Involves Two Distinct Regions of the Cytoplasmic Tail A ROLE FOR RESIDUES ON THE HYDROPHOBIC FACE OF A PUTATIVE AMPHIPATHIC HELIX

Following agonist stimulation, many receptors are rapidly internalized from the plasma membrane via a mechanism which presumably involves recognition motifs within the cytoplasmic domains of the receptor. We have previously demonstrated (Thomas, W. G., Thekkumkara, T. J., Motel, T. J., and Baker, K. M. (1995) J. Biol. Chem. 270, 207-213) that truncation of the angiotensin II (AT1A) receptor, to remove 45 amino acids from the cytoplasmic tail, markedly reduced agonist stimulated receptor endocytosis. In the present study, we have stably and transiently expressed wild type and carboxyl terminus mutated AT1A receptors in Chinese hamster ovary cells to identify regions and specific amino acids important for this process. Wild type AT1A receptors rapidly internalized (t = 2.5 min; Ymax = 76.4%) after AII stimulation. Using AT1A receptor mutants, truncated and deleted at the carboxyl terminus, two distinct regions important for internalization were identified: one membrane proximal site between residues 315-329 and another distal to Lys333, within the terminal 26 amino acids. Point mutations (Y302A, Y312A, L316F, Y319A, and K325A) were performed to identify residues contributing to the membrane proximal site. Mutation of Y302A, Y312A, and K325A had little effect on the rate (t = 4.3, 2.8, and 2.8 min) and maximal amount (Ymax = 81.7, 67.8, and 73.5%) of AII induced internalization. In contrast, L316F and Y319A mutations displayed an approximately 2.5-fold reduction in rate (t = 6.1 and 6.2 min) and L316F a decreased maximal level (Ymax = 38.1 and 71.4%, respectively) compared to wild type. Interestingly, Leu316 and Tyr319 are closely aligned within the hydrophobic aspect of a putative amphipathic helix, possibly representing an internalization motif for the AT1A receptor. We conclude that the AT1A receptor does not use the NPXXY (NPLFY302) motif, first described for the β2-adrenergic receptor, to mediate agonist stimulated endocytosis. Rather, two distinct regions of the carboxyl terminus are utilized: one involving hydrophobic and aromatic residues on a putative α-helix and another serine/threonine-rich domain.

A Functional Role for Sodium-Dependent Glucose Transport across the Blood-Brain Barrier during Oxygen Glucose Deprivation

In the current study, we determined the functional significance of sodium-dependent/-independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter (GLUT) 1 and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [3H]d-glucose and also by [14C]α-methyl d-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT. In vivo middle cerebral artery occlusion experiments were tested to determine whether blood-brain barrier (BBB) SGLT activity was induced during ischemia. Increases in luminal d-glucose and AMG uptake and transport were observed with in vitro stroke conditions. Specific inhibitor experiments suggest a combined role for both SGLT and GLUT1 at the BBB during OGD. A time-dependent increase in the uptake of AMG was also seen in mice exposed to permanent focal ischemia, and this increase was sensitive to the SGLT inhibitor, phlorizin. Infarct and edema ratio during ischemia were significantly decreased by the inhibition of this transporter. These results show that both GLUT1 and SGLT play a role at the BBB in the blood-to-brain transport of glucose during ischemic conditions, and inhibition of SGLT during stroke has the potential to improve stroke outcome. Pharmacological modulation of this novel BBB transporter could prove to be a brain vascular target in stroke.

Regulation of Megalin Expression in Cultured Proximal Tubule Cells by Angiotensin II Type 1A Receptor- and Insulin-Mediated Signaling Cross Talk

Impairment of proximal tubular endocytosis of glomerular-filtered proteins including albumin results in the development of proteinuria/albuminuria in patients with chronic kidney disease. However, the mechanisms regulating the proximal tubular function are largely unknown. This study aimed to investigate the role of angiotensin II type 1A receptor (AT(1A)R)- and insulin-mediated signaling pathways in regulating the expression of megalin, a multiligand endocytic receptor in proximal tubule cells (PTCs). Opossum kidney PTC-derived OK cells that stably express rat AT(1A)R but are deficient in endogenous angiotensin II receptors (AT(1A)R-OK cells) were used for this study. Treatment of the cells with angiotensin II suppressed mRNA and protein expression of megalin at 3- and 24-h incubation time points, respectively. Cellular uptake and degradation of albumin and receptor-associated protein, megalins endocytic ligands were suppressed 24 h after angiotensin II treatment. The AT(1A)R-mediated decrease in megalin expression was partially prevented by ERK inhibitors. Insulin competed with the AT(1A)R-mediated ERK activation and decrease in megalin expression. Inhibitors of phosphatidylinositol 3-kinase (PI3K), a major component of insulin signaling, also suppressed megalin expression, and activation of the insulin receptor substrate (IRS)/PI3K system was prevented by angiotensin II. Collectively the AT(1A)R-mediated ERK signaling is involved in suppressing megalin expression in the OK cell line, and insulin competes with this pathway. Conversely, the insulin-IRS/PI3K signaling, with which angiotensin II competes, tends to stimulate megalin expression. In conclusion, there is AT(1A)R- and insulin-mediated competitive signaling cross talk to regulate megalin expression in cultured PTCs.

Proceedings of the Symposium ‘Angiotensin AT1 Receptors: From Molecular Physiology to Therapeutics’: MOLECULAR MECHANISMS OF ANGIOTENSIN II (AT1a) RECEPTOR ENDOCYTOSIS

1 Angiotensin II (AngII) initiates a variety of cellular responses through activation of type 1 (AT1; with subtypes AT1a and AT1b) and type 2 (AT2) cell surface angiotensin receptors. Both AT1 and AT2 receptors couple to heterotrimeric guanyl nucleotide binding proteins (G‐proteins) and generate intracellular signals following recognition of extracellular AngII, but only AT1 is targeted for the rapid ligand‐stimulated endocytosis (internalization) typical of many plasma membrane receptors. 2 AT1 endocytosis proceeds through clathrin‐coated pits and is independent of G‐protein coupling which predicts that the AngII‐AT1 receptor complex attains a conformation necessary for interaction with the endocytotic machinery, but separate from receptor signalling activation. 3 The function of AT1 endocytosis and the reason for the disparity between AT1 and AT2 endocytosis is not fully appreciated, but the latter probably reflects differences in the primary amino acid sequence of these two receptor types. 4 For many receptors that undergo internalization, it has been established that internalization motifs (2–6 amino acids, often incorporating crucial tyrosine and hydrophobic amino acids) within the cytoplasmic regions of the receptor mediate the selective recruitment of activated receptors into clathrin‐coated pits and vesicles. 5 Mutagenesis studies on the AT1a receptor, aimed at identifying such motifs, reveal that sites within the third cytoplasmic loop and the cytoplasmic carboxyl terminal region are important for AngII‐stimulated AT1a receptor endocytosis.

Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1A are present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood, because specific binding of ANG II to intact PTEC has not been found and because a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells, which stably express AT1A( K d = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of nontransfected OK cells revealed no evidence of AT1A mRNA (reverse transcriptase-polymerase chain reaction analysis) or protein (125I-labeled ANG II binding studies) expression. In cells stably expressing AT1A, ANG II binding was saturable, reversible, and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions that allowed selective access to AP or BL surfaces. AT1A were expressed on both AP ( K d = 8.7 nM, Bmax = 3.33 pmol/mg protein) and BL ( K d = 10.1 nM, Bmax = 5.50 pmol/mg protein) surfaces. Both AP and BL AT1Areceptors underwent agonist-dependent endocytosis (AP receptor: t 1/2 = 7.9 min, Ymax = 78.5%; BL receptor: t 1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, ANG II caused time- and concentration-dependent increases in transepithelial22Na transport (2-fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line that expresses AT1A on both AP and BL surfaces, undergoes agonist-dependent receptor endocytosis, and is functional, as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC.

2-Methoxyestradiol binding of GPR30 down-regulates angiotensin AT1 receptor

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [(3)H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells.

G-protein coupled Angiotensin II receptors (AT1A), mediate cellular responses through multiple signal transduction pathways. In AT1A receptor-transfected CHO-K1 cells (T3CHO/AT1A), angiotensin II (AII) stimulated a dose-dependent (EC50=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT1, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca2+/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [3H]thymidine incorporation in T3CHOA/AT1A cells. AII (1 μM) significantly inhibited cell number (51% at 96 h) and [3H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT1 receptor. Forskolin (10 μM) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [3H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.

Evidence against a role for protein kinase C in the regulation of the angiotensin II (AT1A) receptor

Three putative protein kinase C phosphorylation sites in the carboxyl-terminal region of the angiotensin II AT1A receptor suggest that protein kinase C is involved in the regulation and desensitisation of this receptor. We investigated this possibility by measuring angiotensin II induced Ca2+ transients in cultures of neonatal rat cardiac fibroblasts which express predominantly the angiotensin AT1A receptor. Stimulating or inhibiting protein kinase C activity had no effect on angiotensin II stimulated Ca2+ transients. In addition, in situ and in vitro kinase assays revealed that a peptide, corresponding to the region of the angiotensin AT1A receptor containing the protein kinase C sites, was a poor substrate for protein kinase C. Thus, a heterologous desensitising role for this kinase on angiotensin AT1A receptors in these fibroblasts appears unlikely.

Pharmacologic Effects of 2-Methoxyestradiol on Angiotensin Type 1 Receptor Down-Regulation in Rat Liver Epithelial and Aortic Smooth Muscle Cells

BACKGROUND Delayed onset of cardiovascular disease (CVD) in female patients is not well understood, but could be due in part to the protective effect of estrogen before menopause. Experimental studies have identified the angiotensin type 1 receptor (AT1R) as a key factor in the progression of CVD. OBJECTIVE We examined the effects of the estrogen metabolite 2-methoxyestradiol (2ME2) on AT1R expression. METHODS Rat liver cells were exposed to 2ME2 for 24 hours, and angiotensin II (AngII) binding and AT1R mRNA expressions were assessed. RESULTS In the presence of 2ME2, cells exhibited significant down-regulation of AngII binding that was both dose and time dependent, independent of estrogen receptors (ERα/ERβ). Down-regulation of AngII binding was AT1R specific, with no change in receptor affinity. Under similar conditions, we observed lower expression of AT1R mRNA, significant inhibition of AngII-mediated increase in intracellular Ca(2+), and increased phosphorylation of ERK1/2. Pretreatment of cells with the MEK inhibitor PD98059 prevented 2ME2-induced ERK1/2 phosphorylation and down-regulation of AT1R expression, which suggests that the observed inhibitory effect is mediated through ERK1/2 signaling intermediates. Similar analyses in stably transfected CHO (Chinese hamster ovary) cell lines with a constitutively active cytomegalovirus promoter showed no change in AT1R expression, which suggests that 2ME2-mediated effects are through transcriptional regulation. The effects of 2ME2 on AT1R down-regulation through ERK1/2 were consistently reproduced in primary rat aortic smooth muscle cells. CONCLUSIONS Because AT1R has a critical role in the control of CVD, 2ME2-induced changes in receptor expression may provide beneficial effects to the cardiovascular and other systems.

Delivery of NADPH-cytochrome P450 reductase antisense oligos using avidin-biotin approach.

Although avidin-mediated intracellular delivery of oligonucleotides or proteins has been shown before, the efficacy studies are lacking. Here, we tested the effectiveness of avidin for delivery of a cytochrome P450 reductase (CPR) antisense oligo in rat liver epithelial cells. A phosphorodiamidate morpholino oligo (PMO) against CPR was biotinylated using four reagents with short, cleavable, or long linkers, followed by conjugation with avidin. The dose-inhibitory response of the unmodified PMO in the presence of a transfection reagent (Endoporter, EP) and the effectiveness of the EP-assisted and avidin-assisted delivery of biotinylated PMOs were tested by Western blot analysis. Additionally, in a preliminary study, the avidin-biotin PMO with a long linker was also tested in vivo in rats. The biotinylated oligos were at least as effective as the unmodified oligo. Whereas the avidin conjugate of biotinylated PMO with the short linker was ineffective, those with the long linkers showed significant reductions in CPR protein expression. Finally, the in vivo study showed modest, but significant, reductions in CPR activity. In conclusion, these studies show for the first time that avidin-mediated intracellular delivery of biotinylated oligos can effectively knock down target genes in vitro, depending on the length of the linker. Additionally, the avidin-biotin approach may be of potential value for in vivo gene knockdown.