UMST, Phosphoinositide-calcium signaling in cell regulation The Unit of Molecular Signal Transduction directed by Tamas Balla investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases. Phosphorylation of phosphatidylinositol (PI) to PI 4-phosphate is one of the key reactions in the production of phosphoinositides, lipid regulators of several cellular functions. This reaction is catalyzed by multiple enzymes that belong to either the type-II or the type-III family of PI 4-kinases based on the distinct catalytic properties of the two groups of enzymes. Type-III enzymes are structurally similar to PI 3-kinases and are sensitive to PI 3-kinase inhibitors, and two such enzymes have been cloned and characterized. Relatively little is known about the functions of the type-II PI4Ks in spite of extensive biochemical studies demonstrating their presence in a number of membrane compartments and organelles. The molecular identity of the type-II enzyme was recently revealed when the enzyme was cloned, based on purification of the protein from secretory granules by Barylko et al. (2001). Because of its dissimilarity to any known lipid or protein kinases, the type-II PI 4-kinase enzyme defines a novel enzyme family. By searching the database for homologues of the recently published type-II PI4K, we identified a protein sequence with significant homology with the type-II PI4K enzyme. We termed this sequence PI 4-kinase type-IIbeta, to distinguish it from the previously cloned type-IIalpha enzyme. Based on Northern analysis, transcripts for both enzymes showed a relatively uniform tissue distribution with only a few notable differences, such as the prominent abundance of type-IIbeta, but not type-IIa, mRNA in liver, and the relatively low level of type-IIbeta mRNA in the brain and peripheral leukocytes. Characterization of the catalytic activities of the two enzymes showed that both enzymes are bona fide type-II PI 4-kinases, but type-IIbeta was less active than type-IIalpha. Analysis of the intracellular distribution of the two enzymes revealed prominent association of both enzymes with the endosomal compartments in COS-7 cells. In addition, a significant amount of type-IIalpha, but only a small fraction of type-IIbeta, was associated with the plasma membrane. Catalytically inactive mutant forms of the enzymes showed more prominent plasma membrane localization and the accumulation of numerous vesicles in the juxtanuclear region of the cell, especially in the case of the type-IIa form. In addition, small tubular structures were observed in some of the cells expressing high level of the kinase-inactive proteins, which were much more pronounced in the case of the inactive type-IIbeta enzyme. Co-localization experiments have shown that during trafficking of both the nutrient transferrin receptor and the G protein-coupled AT1 angiotensin receptor passes through endosomes positive for the type-II PI 4-kinase isoforms. Expression of the kinase-inactive forms of the proteins was found to interfere with the rate of transferrin receptor endocytosis, indicating a functional role of these enzymes in the endocytic process. Activation of G protein-coupled receptors (GPCRs) results in the sequestration of the receptors in clathrin-coated pits in the plasma membrane, followed by endocytosis of the ligand-receptor complex. After endocytosis of most GPCRs, the majority of the receptor is recycled back to the cell surface but some of the receptor and most of the ligand is degraded via lysosomal or proteosomal degradative pathways. Little is known about the pathways involved in GPCR trafficking, and even less about the molecular mechanisms directing the receptors toward the various vesicular trafficking routes. The involvement of phosphoinositides at numerous steps in vesicular trafficking is well documented in all species from yeast to mammals. To study AT1-R trafficking and the role of phosphoinositides, we created stable cell lines of HEK 293 cells expressing either a HA-tagged AT1-R receptor or an AT1-R with a GFP molecule fused to its C-terminus. To follow the ligand, we employed Rhodamine-labeled angiotensin II and identified the various endocytic compartments by using GFP-fused Rab proteins or GFP-fused protein domains that recognize PI(3)P. After stimulation with Ang II, the receptor and its ligand co-localized with rab5 and rab4- in early endosomes, and subsequently with rab11 in pericentriolar recycling endosomes. Inhibition of PI 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogenous Rab composition, containing the ligand-receptor complex in their limiting membranes, and in small vesicular structures associated with the large vesicles. In wortmannin-treated cells, Alexa-transferrin was found in small vesicles associated exclusively with the outside of large vesicles, while Rhod-Ang II was also segregated into small vesicles in the lumen of the larger endosomes. Wortmannin treatment did not affect the late appearance of either Alexa-transferrin or Rhod-Ang II in pericentriolar recycling endosomes. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early but not the slow component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. Our data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors and proceeds via the pericentriolar recycling endosomal compartment.
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