Research Projects
Our research program is in the areas of signal transduction and membrane trafficking. It is aimed at elucidating at the molecular level the pathways of signaling from the insulin receptor and the trafficking of membrane proteins to the cell surface in response to insulin.
Signaling
In our earlier research on insulin signaling we purified, cloned, and characterized the insulin receptor substrates 1, 3, and 4 (IRS-1,3,4). These three proteins, together with a fourth member of the family known as IRS-2, play a key role in insulin signaling. They act as docking/effector proteins for a number of SH2 domain-containing signaling proteins and thereby link the insulin receptor to the signaling pathways downstream of it.
Our current research on insulin signaling has two directions. First, we plan to determine the insulin phosphoproteome, which we define as all the sites on all the proteins that undergo an acute change in phosphorylation in response to insulin. Insulin treatment of cells rapidly changes the activity of a number of kinases and phosphatases, leading to changes in the extent of phosphorylation on many sites on many proteins. We plan to determine the insulin phosphoproteome in cultured fat, muscle, and liver cells, which are models for the main target tissues of insulin action. New advances in mass spectrometry have made the determination of the insulin phosphoproteome possible. This project is a joint one with Dr. Scott Gerber, an expert in mass spectrometry and phosphoproteomics, who is also at Dartmouth Medical School. We estimate that the number of sites that change in response to insulin is 1600, of which we currently know about 400. Hence, a large portion of the insulin phosphoproteome is not known, and the results of this project will greatly expand our knowledge of insulin signaling.
Second, we are investigating the molecular and cellular effects of insulin-stimulated phosphorylation of several novel proteins that we have found by more targeted phosphoproteomic analyses. One of these proteins is a GTPase activating protein (GAP) for the small G protein Ral, known as Ral-GAP-A2. We discovered that Ral-GAP-A2 is phosphorylated on several sites by the insulin-activated protein kinase Akt (also known as protein kinase B). We are now determining whether this phosphorylation inhibits the activity of Ral-GAP-A2, and so accounts for the known effect of insulin to increase the level of the GTP form of Ral in cells. Ral in its GTP form participates in vesicle docking to the plasma membrane (see below) as well as in the regulation of gene expression and mRNA translation.
Membrane Trafficking
Insulin lowers the level of blood glucose in part by stimulating the transport of glucose into fat and muscle cells. The basis for this stimulation is an increase in the number of glucose transporters in the plasma membrane. This increase is due to the insulin-stimulated docking and then fusion of specialized vesicles containing glucose transporters with the plasma membrane. The glucose transporter isotype present in fat and muscle cells is known as GLUT4. Hence the intracellular vesicles are known as GLUT4 vesicles, and overall process is known as GLUT4 translocation.
In our earlier research we purified GLUT4 vesicles and analyzed their protein content. Among the proteins that we found are: Rab10, a small G protein that participates in vesicle docking to membranes; VAMP-2, a vesicular SNARE protein that participates in the fusion of docked vesicles; and a novel membrane aminopeptidase. Subsequent studies from our lab and others have implicated Rab10 and VAMP-2 in the insulin-stimulated docking and fusion of GLUT4 vesicles, respectively. The aminopeptidase is a vesicle cargo protein that exhibits trafficking behavior identical to that of GLUT4. Thus, insulin increases the aminopeptidase activity, as well as the glucose transport activity, at the surface of the cell.
We currently are focused on two goals for our research on GLUT4 translocation. One is to elucidate the connections between insulin signaling and GLUT4 translocation at the molecular level. The other is to describe how Rab10 functions in GLUT4 translocation at the molecular level.
With regard to the first goal, we have discovered a GAP for Rab10 known AS160 (also known as TBC1D4) and found that AS160 is phosphorylated on multiple sites by the insulin-activated protein kinase Akt. Current evidence indicates that phosphorylation of AS160 suppresses its GAP activity, which thereby results in an elevation of the GTP form of Rab10 on the GLUT4 vesicles. The latter then triggers vesicle docking to the plasma membrane. A protein complex known as the exocyst participates in docking of GLUT4 vesicles to the plasma membrane. We and others have found that Rab10 in its GTP form associates with the exocyst. Assembly of the functional exocyst complex also requires the GTP form of Ral. Hence, if the insulin-stimulated phosphorylation of Ral-GAP-A2 accounts for the insulin-stimulated elevation in the GTP form of Ral (see above), this process would be a second signaling input, in addition to the phosphorylation of AS160, into GLUT4 translocation. Finally, there is evidence that insulin stimulates the fusion of docked GLUT4 vesicles with the plasma membrane. The signaling input for this step is not known. We are searching for a molecular link between insulin signaling and the proteins of the fusion machinery.
With regard to the second goal, we have conducted a yeast two-hybrid screen with constitutively active Rab10 and identified a number of interacting proteins. We are attempting to determine which of these interacting proteins participates in GLUT4 translocation, and how those that participate do so. One of the interacting proteins is a subunit of the exocyst complex, and a current project is to determine whether this interaction is required for docking of the GLUT4 vesicles to the plasma membrane.
Relevance to Disease
The basic science that we are doing is very relevant to diabetes. Approximately 15,000,000 Americans suffer from adult-onset diabetes, also referred to as type 2 diabetes and non-insulin dependent diabetes. A major feature of this disease is that insulin has reduced effectiveness in its action upon its target tissues of muscle, fat, and liver, including reduced GLUT4 translocation in muscle and fat. This feature is referred to as insulin resistance. The basis for insulin resistance is not understood. Fundamental research of the type herein provides the framework for identification of the changes that cause insulin resistance in type 2 diabetes, as well as leading to potential targets for therapeutic agents.