Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell- to-cell communication. RTKs are normally under tight regulatory control and have low basal (non-ligand bound) activity; they are activated transiently in response to specific stimuli. Insulin and insulin-like growth factor-1 (IGF1) are closely related polypeptide hormones/growth factors that regulate cell growth and metabolism. Insulin and IGF1 exert their biological effects on target cells by binding to distinct cell surface receptors, the insulin receptor (IR) and the IGF1 receptor (IGF1R), respectively, which are structurally related RTKs. Despite extensive structural and biochemical studies of the separate ectodomains and cytoplasmic domains of these receptors, a comprehensive understanding of the signal transduction mechanism for IR and IGF1R is still lacking. To address these issues, we have produced highly purified preparations of full-length IR and IGF1R that are detergent-soluble, functional, and responsive to their ligands. By complexing the receptors with known downstream signaling proteins such as tyrosine phosphatase PTP1B and adapter protein IRS1, we will determine by single-particle cryo-electron microscopy the configurations of the kinase domains and downstream proteins as well as the specificity factors governing recruitment of these proteins to the activated receptors. We will carry out functional mutagenesis experiments in mammalian cells to confirm structural results, and to probe for sites of allosteric regulation. We will test IGF1R mutations recently found to be associated with type 2 diabetes. Using a computational method (virtual ligand screening), we will identify novel small molecule IR activators. We will also test the hypothesis that cellular sterol and lipid composition affects transmembrane signaling by IR and IGF1R. To do this, we will take advantage of new methodology to manipulate the composition of the plasma membrane in living cells. In our preliminary work, we have used this approach to demonstrate the importance of membrane sterols on IR and IGF1R signaling. Our work supports the hypothesis that IR localizes in liquid-ordered ("raft") domains when active. We will test this hypothesis via deletion or insertion of hydrophobic residues. We will examine the possibility that statins alter the plasma membrane environment and lead to decreased IR function. We will also reconstitute IR into lipid vesicles with and without raft domains, and use fluorescence resonance energy transfer (FRET) to determine whether IR moves to rafts upon insulin stimulation. The work proposed in this grant application will advance our knowledge of the molecular mechanisms involved in transmembrane signaling by IR and IGF1R. This information should prove to be valuable in the design of small-molecule agonists or inhibitors to modulate the function of these RTKs.