Structural Studies of Receptor Tyrosine Kinases

Many important cellular signaling cascades are initiated at the cell surface by the binding of a polypeptide ligand to a transmembrane receptor possessing intrinsic tyrosine kinase activity in its cytoplasmic domain. The receptor tyrosine kinase (RTK) family includes, among others, the insulin receptor, insulin-like growth factor-1 (IGF1) receptor, epidermal growth factor receptor, fibroblast growth factor receptor and MuSK, the receptor for agrin. Ligand binding induces receptor oligomerization (growth factor receptors) or a conformational change within the receptor (insulin/IGF1 receptor), leading to autophosphorylation of specific tyrosine residues in the cytoplasmic domains of the receptors. Tyrosine autophosphorylation stimulates receptor catalytic activity and generates recruitment sites for downstream signaling proteins. RTKs are critical components in signal transduction pathways that mediate cell proliferation, differentiation, migration and metabolism, and are active during organismal development and adult homeostasis. RTKs also play primary roles in the onset or progression of pathological conditions such as diabetic retinopathy, atherosclerosis and cancer.

Insulin Receptor
Using x-ray crystallography as our primary experimental technique, we are attempting to understand the molecular basis for insulin receptor activation and for recruitment of downstream signaling proteins to the activated (phosphorylated) insulin receptor. Several cytoplasmic adapter proteins bind to the activated insulin receptor, including insulin receptor substrate (IRS) proteins and APS, which are positive factors in insulin signaling pathways culminating in glucose uptake. The insulin receptor is downregulated by the adapter proteins Grb10 and Grb14 as well as the tyrosine phosphatase PTP1B. We are determining crystal structures of complexes between these proteins and the insulin receptor kinase domain to elucidate the modes of interaction and the determinants of specificity.

The KRLB region of IRS2 bound to tris-phosphorylated IRK. The N-terminal kinase lobe is colored dark gray, the C-terminal lobe is colored light gray, and the KRLB region (residues 620-634) is shown in stick represenetation. Atoms of the activation loop and catalytic loop of IRK are colored green and orange, respectively. [Wu et al., Nat. Struct. Mol. Biol. (in press, 2008)]

The BPS region of Grb14 bound to tris-phosphorylated IRK. The N-terminal kinase lobe is colored dark gray, the C-terminal lobe is colored light gray, and the BPS region is colored purple. The activation loop and catalytic loop of IRK are colored green and orange, respectively. [Depetris et al., Mol. Cell 20, 325-333 (2005)]

APS SH2 dimer bound to tris-phosphorylated IRK. The two APS(SH2) protomers are colored orange and blue, and the two IRK molecules are shown with a semi-transparent surface. [Hu et al., Mol. Cell 12, 1379-1389 (2003)]

IGF1 Receptor
The IGF1 receptor is highly related in sequence and structure to the insulin receptor, but has distinct biological functions, one of which is cell survival. Therefore, this RTK is a potential target for inhibition in tumor cells. In collaboration with Dr. Todd Miller at SUNY-Stony Brook, we have determined the three-dimensional structure of the IGF1 receptor kinase domain using x-ray crystallography. Several amino acid differences between the IGF1 receptor and the insulin receptor near the ATP binding cleft might be exploited by small-molecule inhibitors to gain selectivity for the IGF1 receptor over the insulin receptor. To this end, structural studies of inhibitors bound to the IGF1 receptor kinase are being pursued.

Surface representation of the IGF1 receptor tyrosine kinase domain. The bound ATP analog and substrate peptide are shown in stick representation. Colored green and yellow are the residues that differ between the IGF1 receptor and the insulin receptor. [Favelyukis et al., Nat. Struct. Biol. 18, 1058-1063 (2001)]

MuSK
Another RTK of interest is MuSK, or muscle-specific kinase, which is expressed exclusively in muscle cells and plays an essential role in the formation of neuromuscular synapses by promoting clustering of acetylcholine receptors. Activation of MuSK by agrin results in autophosphorylation of several tyrosines in the cytoplasmic domain of MuSK. In a collaboration with Dr. Steven Burden at the NYU School of Medicine, we have determined the crystal structure of the cytoplasmic (tyrosine kinase-containing) domain of MuSK to understand how kinase activity is regulated in this receptor. The structure reveals that MuSK is strongly autoinhibited by the kinase activation loop and suggests that an additional in vivo component might contribute to negative regulation by binding to the juxtamembrane region of MuSK.

Ribbon diagram of the tyrosine kinase domain of MuSK. Beta strands are colored cyan and alpha helices are colored red/yellow. The juxtamembrane region and activation loop are colored green. The activation loop contains three tyrosine autophosphorylation sites whose side chains are colored black. Disordered regions of the structure are represented as dashed lines. [Till et al., Structure 10, 1187-1196 (2002)]

We have also determined the crystal structure of the first two immunoglobulin-like domains (Ig1-2) of the MuSK extracellular region. These domains are critical for activation of the receptor by agrin. Ig1-2 crystallized as a dimer, mediated by Ig1, and the residues in the dimer interface are critical for agrin-induced phosphorylation of the receptor. Whether these residues are important for receptor dimerization or for a heterologous interaction (e.g., with agrin or a co-receptor) is still under investigation. 

Molecular surface representation of dimeric Ig1-2 of the MuSK ectodomain. Ig1 is colored light green/purple, and Ig2 is colored dark green/purple. The dimer interface is mediated solely by residues in Ig1. [Stiegler et al., J. Mol. Biol. 364, 424-433 (2006)]