We investigate 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 domain and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in living cells; and (5) determining the importance of lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.

Characterization of a New Member of the Type-II Phosphatidylinositol 4-Kinase Family
Balla A, Tuymetova, Barshishat, Balla T
Phosphorylation of phosphatidylinositol (PI) to PI 4-phosphate is one of the key reactions in the production of phosphoinositides, which are lipid regulators of several cellular functions. The reaction is catalyzed by multiple enzymes that belong to either the type-II or type-III family of PI 4-kinases depending on the distinct catalytic properties of the two groups of enzymes. Type-III enzymes are structurally similar to PI 3kinases 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. at the University of Texas Southwestern Medical Center. 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 homologs of the recently published type-II PI4K, we identified a protein sequence with significant homology to the type-II PI4K enzyme. We termed this sequence PI 4-kinase type-IIb in order to distinguish it from the previously cloned type-IIa 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-IIb, but not type-IIa, mRNA in liver, and the relatively low level of type-IIb 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-IIb was less active than type-IIa. Analysis of the intracellular distribution of the two enzymes in COS-7 cells revealed prominent association of both enzymes with the endosomal compartments. In addition, a significant amount of type-IIa, but only a small fraction of type-IIb, was associated with the plasma membrane (Fig. 6).

Figure 6

Cellular distribution of PI 4-kinase type-IIa and type-IIb (upper panels) or their kinase-inactive mutant forms D308A and D304A of type-IIa and IIb (lower panels), respectively, expressed in COS-7 cells as GFP fusion proteins. Confocal images; bar represents 10 mm.

Catalytically inactive mutant forms of the enzymes (kinase-inactive proteins) 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 a high level of the kinase-inactive proteins, which were much more pronounced in the case of the inactive type-IIb enzyme. Co-localization experiments have shown that during their trafficking both the nutrient transferrin receptor and the G protein–coupled AT angiotensin receptor pass through endosomes positive for 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 the enzymes in the endocytic process.

Trafficking Pattern of Internalized AT1 Angiotensin Receptors: Identification of PI 3-Kinase–Dependent and –Independent Pathways during AT1 Receptor Recycling
Balla T; in collaboration with Hunyady, Baukal, Gàborik , Lodge, Catt
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, most of the receptor is recycled back to the cell surface, although some of the receptor and most of the ligand are 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.

Figure 7

Internalization of AT1 angiotensin receptors in HEK 293 cells stably expressing an AT1-R-GFP chimeric construct. Confocal images of cells stimulated by angiotensin II for the indicated times.

To study AT1-R trafficking and the role of phosphoinositides, we created stable HEK 293 cell lines 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 (Rhod-Ang 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 heterogeneous Rab composition, which contained the ligand-receptor complex in their limiting membranes and in small vesicular structures associated with the large vesicles. In WT-treated cells, Alexa-transferrin (transferrin labeled with a different fluorophore) was found in small vesicles associated exclusively with the outside of large vesicles, while Rhod-Ang II also segregated into small vesicles in the lumen of the larger endosomes. WT treatment did not affect the late appearance of either Alexa-transferrin or Rhod-Ang II in pericentriolar recycling endosomes. In cells labeled with 125IAng II, WT treatment did not impair the rate of receptor endocytosis, but it 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 that proceeds via the pericentriolar recycling endosomal compartment.