We have determined the 3.2 A X-ray crystal structure of the extracellular domain of the human epidermal growth factor receptor 2 (ErbB2 or HER2) in a complex with the antigen binding fragment of pertuzumab, an anti-ErbB2 monoclonal antibody also known as 2C4 or Omnitarg. Pertuzumab binds to ErbB2 near the center of domain II, sterically blocking a binding pocket necessary for receptor dimerization and signaling. The ErbB2-pertuzumab structure, combined with earlier mutagenesis data, defines the pertuzumab residues essential for ErbB2 interaction. To analyze the ErbB2 side of the interface, we have mutated a number of residues contacting pertuzumab and examined the effects of these mutations on pertuzumab binding and ErbB2-ErbB3 heterodimerization. We have also shown that conserved residues previously shown to be necessary for EGF receptor homodimerization may be dispensible for ErbB2-ErbB3 heterodimerization.

We show that two commonly occurring epidermal growth factor receptor (EGFR) somatic mutations, L858R and an in-frame deletion mutant, Del(746-750), exhibit distinct enzymatic properties relative to wild-type EGFR and are differentially sensitive to erlotinib. Kinetic analysis of the purified intracellular domains of EGFR L858R and EGFR Del(746-750) reveals that both mutants are active but exhibit a higher K M for ATP and a lower K i for erlotinib relative to wild-type receptor. When expressed in NR6 cells, a cell line that does not express EGFR or other ErbB receptors, both mutations are ligand dependent for receptor activation, can activate downstream EGFR signaling pathways, and promote cell cycle progression. As expected from the kinetic analysis, the EGFR Del(746-752) is more sensitive to erlotinib inhibition than the EGFR L858R mutant. Further characterization shows that these mutations promote ligand-dependent and anchorageindependent growth, and cells harboring these mutant receptors form tumors in immunocompromised mice. Analysis of tumor lysates reveals that the tumorigenicity of the mutant EGFR cell lines may be due to a differential pattern of mutant EGFR autophosphorylation as compared with wild-type receptor. Significant inhibition of tumor growth, in mice harboring wild-type EGFR receptors, is only observed at doses of erlotinib approaching the maximum tolerated dose for the mouse. In contrast, the growth of mutant tumors is inhibited by erlotinib treatment at approximately one third the maximum tolerated dose. These findings suggest that EGFR somatic mutations directly influence both erlotinib sensitivity and cellular transformation. (Cancer Res 2006; 66(16): 8163-71)

Mer/Nyk/Eyk is an orphan receptor tyrosine kinase expressed at high levels in monocytes and cells derived from epithelial and reproductive tissues. Overexpression of Mer has been associated with lymphoid malignancies. Here we identify Gas6, the product of a growth arrest speci®c gene, as a ligand for Mer. Gas6 has previously been shown to activate both Axl and Rse/Tyro3, two other receptor tyrosine kinases in the same family as Mer. The apparent relative association and dissociation rate constants of Gas6 for soluble Axl, Rse/Tyro3 and Mer were compared using surface plasmon resonance. Gas6 was shown to induce rapid phosphorylation of Mer expressed in several dierent types of cells. We also observed a transient activation of p42 MAP kinase following activation of Mer by Gas6. Thus, Gas6 exerts its biological eects through multiple receptor tyrosine kinases.

We used the yeast two-hybrid system to identify proteins that interact directly with G␣ o . Mutant-activated G␣ o was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that G␣ o interacted with several proteins including Gz-GTPaseactivating protein (Gz- Heterotrimeric G proteins function as signal transducers for receptors for a large number of hormones, neurotransmitters, autocrine and paracine factors, cytokines, and sensory signals. Both the G␣ (1) and the G␤␥ subunits (2) are capable of transferring receptor signals to effectors. There are four families of G␣ subunits (3). Direct effectors for the G␣ s (4) and G␣ q (5) family proteins have been well characterized. Effectors for the G␣ i family are less well defined. G␣ t , the visual G protein, activates the cGMP phosphodiesterase (1), and G␣ i inhibits adenylyl cyclases (6) directly (7). However, direct effectors for G␣ o , an abundant G protein in the brain (8, 9), have not yet been identified. G␣ o has been implicated in receptor-mediated inhibition of Ca 2ϩ channels in chick dorsal root ganglion neurons (10). Hence, it appeared feasible that this system could be used to identify proteins that directly interact with G␣ o . We used the yeast two-hybrid system to identify potential G␣ o effectors. For this purpose, we screened the chick dorsal root ganglion cDNA library with the mutationally (Q205L) activated form of G␣ o . In this article we present data indicating that the inactive form of G␣ o preferentially interacts with Rap1GAP 1 and thus regulates the activity of the small G protein Rap.GAP EXPERIMENTAL PROCEDURESMaterials-The cDNA synthesis system was from Life Technologies, Inc. Anti-G␣ o and anti-MAPK2 antibodies were from Santa Cruz Biotechnology, Anti-M2-FLAG antibody was from Sigma, anti-G␣ I-3/o antibody was from Upstate Biotechnology, Inc., and phospho-specific and total MAPK antibodies were from New England Biolabs. Most biochemicals were from Sigma, and cell culture supplies were from Life Technologies, Inc. All restriction enzymes were from New England Biolabs. Yeast culture media and amino acids were from CLONTECH. DNA plasmid preparation reagents and Effectene and Superfect transfection reagents were from Qiagen, Inc. ECL reagents were from Amersham Pharmacia Biotech. All other reagents were of the highest grade available.Yeast Two-hybrid Screening-A directional oligo(dT)-primed cDNA library was constructed from 12-day embryonic chick dorsal root ganglion mRNA. cDNA was synthesized using a cDNA synthesis system and ligated into the GAL4 DNA-activation domain plasmid pPC86 using the SalI/NotI restriction sites (11). Plasmid DNA was isolated from the unamplified library using a Qiafilter Plasmid Maxi Kit. Q205L-G␣ o was cloned into the SalI/NotI restriction sites of the GAL4 DNA-binding domain plasmid pPC97-cycloheximide (11). The Q205L-G␣ o -BD plasmid and the library were co-transformed into the yeast strain MaV203, plated on selective media lacking leucine, tryptophan, and histidine and containi...

Hormones and growth factors regulate cell growth via the mitogen-activated protein (MAP) kinase cascade. Here we examine the actions of the hormone somatostatin on the MAP kinase cascade through one of its two major receptor subtypes, the somatostatin receptor 1 (SSTR1) stably expressed in CHO-K1 cells. Somatostatin antagonizes the proliferative effects of fibroblast growth factor in CHO-SSTR1 cells via the SSTR1 receptor. However, in these cells, somatostatin robustly activates MAP kinase (also called extracellular signal regulated kinase; ERK) and augments fibroblast growth factor-stimulated ERK activity. We show that the activation of ERK via SSTR1 is pertussis toxin sensitive and requires the small G protein Ras, phosphatidylinositol 3-kinase, the serine/threonine kinase Raf-1, and the protein tyrosine phosphatase SHP-2. The activation of ERK by SSTR1 increased the expression of the cyclin-dependent protein kinase inhibitor p21(cip1/WAF1). Previous studies have suggested that somatostatin-stimulated protein tyrosine phosphatase activity mediates the growth effects of somatostatin. Our data suggest that SHP-2 stimulation by SSTR1 may mediate some of these effects through the activation of the MAP kinase cascade and the expression of p21(cip1/WAF1).