The Expression of MTUS1/ATIP and Its Major Isoforms, ATIP1 and ATIP3, in Human Prostate Cancer.

Angiotensin II (Ang II), the main effector of the renin angiotensin system, acts upon two distinct transmembrane receptors, the Ang II type 1 and the type 2 (AT2-) receptor, to induce promotion and inhibition of ERK2 phosphorylation. The AT2-receptor, through an interaction with its putative signaling partner MTUS1/ATIP (AT2-receptor interacting protein), inhibits the mitogenic effects of EGF in prostate cancer cell lines representing both early and late stage disease. This is the first report on the expression of ATIP in normal and malignant human prostatic biopsies. The expression of ATIP and its major isoforms, ATIP1 and ATIP3, in normal prostatic cells and three prostate cancer cell lines was examined using QPCR and immunohistochemistry. Human biopsies containing benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and well, moderately and poorly differentiated prostate cancer were also examined. Overall, ATIP1 and ATIP3 mRNA expression was increased in malignant compared to normal tissues and cell lines. ATIP immunostaining was low or absent in both the basal and columnar epithelial cell layers surrounding BPH acini; however, it was observed in high concentration in neoplastic epithelial cells of HGPIN and was clearly evident in cytoplasms of malignant cells in all prostate cancer grades. ATIP immunostaining was also identified in the cytoplasms of LNCaP and PC3 prostate cancer cells. As the AT2-receptor/ATIP inhibitory signaling pathway exists in malignant cells in all grades of prostate cancer, enhancement of this pathway may be a therapeutic target even after the development of androgen-independence.

Expression and function of ATIP/MTUS1 in human prostate cancer cell lines.

BACKGROUND: We have previously demonstrated Ang II type 2 (AT(2)-) receptor-mediated inhibition of EGF-induced prostate cancer cell growth in androgen-dependent (LNCaP) and independent (PC3) prostate cancer cell lines. METHODS: To explore the signaling pathways involved in this inhibitory effect, we examined the interaction of the AT(2)-receptor with its novel regulatory partner ATIP using real time PCR, over-expression, siRNA and [(3)H]thymidine incorporation assays. RESULTS: The results in human prostate cancer cell lines demonstrate the presence of ATIP in both cell lines examined, and suggest that (i) the AT(2)-receptor through an interaction with ATIP mediates an anti-growth factor effect in both androgen-dependent and androgen-independent cell lines; (ii) ATIP expression decreases as the rate of cell growth and androgen-independence increase; and (iii) EGF may act on cell growth in part by reducing the content of ATIP present in the cells. CONCLUSIONS: The results support our earlier proposal in normal cell lines that ATIP is an important component of the cellular response to AT(2)-receptor activation. The results further suggest that a critical level of ATIP is required to mediate the effect of AT(2)-receptor activation to inhibit EGF mediated increases in cell growth. They also suggest that EGF may in part induce cell growth by suppressing the level of ATIP expression.

Role of Tyr(356(7.43)) and Ser(190(4.57)) in antagonist binding in the rat beta1-adrenergic receptor.

Site-directed mutagenesis and photoaffinity labeling experiments suggest the existence of at least two distinct binding orientations for aryloxypropanolamine competitive antagonists in the beta-adrenergic receptor (beta-AR), one where the aryloxy moiety is located near transmembrane alpha-helix 7 (tm 7) and another where it is near tm 5. To explore a hydrophobic pocket involving tms 1, 2, 3, and 7 for potential aryloxy interaction sites, we selected Tyr(356(7.43)) and Trp(134(3.28)) in the rat beta(1)-AR for site-directed mutagenesis studies. Ser(190(4.57)) was also investigated, as the equivalent residues are known antagonist interaction sites in the muscarinic M(1) and the dopamine D(2) receptors. Binding affinities (pK(i)) of a series of structurally diverse aryloxypropanolamine competitive antagonists were determined for wild type and Y356A, Y356F, W134A, and S190A mutant rat beta(1)-ARs stably expressed in Chinese hamster ovary cells. To visualize possible antagonist/receptor interactions, the compounds were docked into a three-dimensional model of the wild-type rat beta(1)-AR. The results indicate that Tyr(356(7.43)) is an important aromatic interaction site for five of the eight competitive antagonists studied, whereas none of the compounds appeared to interact directly with Trp(134(3.28)). Only two of the competitive antagonists interacted with Ser(190(4.57)) on tm 4. Overall, the results extend our understanding of how beta(1)-AR competitive antagonists bind to the hydrophobic pocket involving tms 1, 2, 3, and 7; highlight the importance of Tyr(356(7.43)) in this binding pocket; and demonstrate the involvement of tm 4 in competitive antagonist binding.

Agonist binding and activation of the rat beta(1)-adrenergic receptor: role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)).

We investigated the role of Trp(134(3.28)), Ser(190(4.57)) and Tyr(356(7.43)) in agonist binding to, and activation of, the rat beta(1)-adrenergic receptor by comparing pK(i)s and functional responses of W134A, S190A and Y356F mutant receptors to wild type, all stably expressed in CHO cells. All three mutations significantly (P < 0.05) reduced adenylyl cyclase intrinsic activity (IA) compared to wild type in response to stimulation with both (-)-isoprenaline (53-88%) and (-)-RO363 (46-61%), and there was no significant correlation either between IA or pD(2) and pK(i) (P > 0.4), suggesting that changes in pK(i) were not sufficient to explain the fall in adenylyl cyclase activity. The most pronounced reduction in affinity (126-fold, P < 0.01) was displayed by xamoterol for the Y356F mutation, suggesting that xamoterol is able to directly interact with Tyr(356(7.43)). For the other agonists, the change in pK(i) values for the mutant receptors ranged from a 20-fold decrease to a 2-fold increase compared to the wild type. In a three-dimensional model of the rat beta(1)-adrenergic receptor, Trp(134(3.28)) and Tyr(356(7.43)) form part of a hydrophobic binding pocket involving residues in transmembrane helices 1, 2, 3 and 7. Our results suggest that Trp(134(3.28)) and Tyr(356(7.43)), together with Trp(353(7.40)), are able to interact via pi-pi interactions to stabilize the extracellular ends of transmembrane helices 3 and 7. Ser(190(4.57)) appears to be involved in a hydrogen bonding network, which maintains the spatial relationship between transmembrane helices 3 and 4. These interhelical interactions suggest that the three mutated residues stabilize the active receptor state by maintaining the proper packing of their respective transmembrane helix within the helix bundle, facilitating the appropriate movement and rotation of the transmembrane regions during the activation process.

Site-directed mutagenesis of the rat beta1-adrenoceptor. Involvement of Tyr356 (7.43) in (+/-)cyanopindolol but not (+/-)[125Iodo]cyanopindolol binding.

To determine the role played by Tyr(356 (7.43)) in the rat beta(1)-adrenoceptor in binding the antagonists (+/-)cyanopindolol (4-[3-(t-butylamino]-3-(2'-cyano-indoloxy)-2-propanolol) and its iodinated analogue (+/-)[(125)Iodo]cyanopindolol (1-(t-butylamino]-3-(2'-cyano-3'-iodo-indoloxy)-2-propanolol), Tyr(356 (7.43)) was mutated to either Phe or Ala and binding affinities determined for wild type and mutant rat beta(1)-adrenoceptors. Our results indicate that Tyr(356 (7.43)) is important for (+/-)cyanopindolol, but not (+/-)[(125)Iodo]cyanopindolol, binding and that (+/-)cyanopindolol adopts a "reverse" binding orientation whereas (+/-)[(125)Iodo]cyanopindolol cannot be accommodated in this binding mode. We define a "reverse" antagonist binding mode as one where the aryloxy moiety interacts with residues on transmembrane helices 1, 2, 3 and 7. The beta(1)-adrenoceptor site-directed mutagenesis results are the first to support a "reverse" antagonist binding orientation and the involvement of Tyr(356 (7.43)) in this binding mode.

Synthesis, beta-adrenoceptor pharmacology and toxicology of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride, a short acting beta(1)-specific antagonist.

The synthesis of S-(-)-1-(4-(2-ethoxyethoxy)phenoxy)-2-hydroxy-3-(2-(3,4-dimethoxyphenyl)ethylamino)propane hydrochloride (D140S.HCl 6), a novel short acting beta(1)-specific adrenoceptor antagonist, has been described. The antagonist potency for D140S.HCl 6 has been compared with esmolol, another short acting agent, and other well known beta-adrenoceptor antagonists in isolated rat tissue preparations. The pharmacokinetics of D140S.HCl 6 in 7 day continuous intravenous infusions and 4 weeks intravenous bolus injection studies in conscious rats and dogs have been examined in toxicology studies. The effect on the isoprenaline-induced heart rate increase and the pharmacodynamic half-life of D140S.HCl 6 has been compared with esmolol in a conscious rat model. In addition, the results of a range of toxicological studies are presented. The results indicate that D140S.HCl 6 is a highly specific beta(1)-adrenoceptor antagonist (pA(2) = 8.15+/-0.22, beta(1)/beta(2) selectivity > 4400). The in vitro studies suggest D140S.HCl is ca. ten times more potent and 60 times more beta(1)-specific than racemic esmolol. Pharmacokinetic non-linearity was seen when given as a 7 day intravenous infusion at toxicological doses above 10 mg kg(-1) h(-1) in the rat and 2.5 mg kg(-1) h(-1) in the dog. Both D140S.HCl 6 and esmolol have very short durations of action after intravenous infusion in the rat (pharmacodynamic half-life is < 15 min for D140S.HCl and 10 min for esmolol). The toxicological tests indicate that D140S.HCl 6 shows no unexpected toxicity and none of the tissue irritancy problems reported for esmolol formulations.