Kathryn M. Ferguson

An Open-and-Shut Case? Recent Insights into the Activation of EGF/ErbB Receptors

Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.

EGF Activates Its Receptor by Removing Interactions that Autoinhibit Ectodomain Dimerization

Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. We describe the 2.8 A resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state. The structure reveals an autoinhibited configuration, where the dimerization interface recently identified in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists.

Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain

The X-ray crystal structure of the high affinity complex between the pleckstrin homology (PH) domain from rat phospholipase C-delta 1 (PLC-delta 1) and inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P3) has been refined to 1.9 A resolution. The domain fold is similar to others of known structure. Ins(1,4,5)P3 binds on the positively charged face of the electrostatically polarized domain, interacting predominantly with the beta 1/beta 2 and beta 3/beta 4 loops. The 4- and 5-phosphate groups of Ins(1,4,5)P3 interact much more extensively than the 1-phosphate. Two amino acids in the PLC-delta 1 PH domain that contact Ins(1,4,5)P3 have counterparts in the Brutons tyrosine kinase (Btk) PH domain, where mutational changes cause inherited agammaglobulinemia, suggesting a mechanism for loss of function in Btk mutants. Using electrostatics and varying levels of head-group specificity, PH domains may localize and orient signaling proteins, providing a general membrane targeting and regulatory function.

PH Domains: Diverse Sequences with a Common Fold Recruit Signaling Molecules to the Cell Surface

With the identification of two distinct classes of high affinity, physiologically relevant, ligands for PH domains, it appears reasonable to assume that additional specific high affinity ligands for other PH domains will be identified in the future. It is not clear, however, whether each of the 90 proposed PH domains will have its own specific ligand. Possible candidates for specific PH domain ligands include various inositol polyphosphates, phosphorylated membrane components, as well as specific protein sequences containing phosphorylated tyrosine, serine, threonine, or histidine residues. It appears unlikely that the low affinity interactions of phosphoinositides described for several PH domains are physiologically relevant. It is difficult to imagine why such a large and diverse family of PH domains (with just 10-15% sequence identity) would exist in order to bind with a similar low affinity to PtdInsP2-containing membranes. Rather, we suggest that these interactions represent limited binding to noncognate ligands - the physiologically relevant ligands have yet to be identified. It is likely that many, if not all, PH domains have their own high affinity, cell membrane-associated, ligands and operate according to the paradigms described for the PH domains of PLCδ1 and Shc (Figure 2Figure 2A and Figure 2Figure 2B). The structural homology between PH domains might reflect a particularly stable protein scaffold of β sheets that can present variable ligand-binding loops in a manner analogous to that seen in the immunoglobulin superfamily.

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3‐kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3‐phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane‐targeted Ras to complement a temperature‐sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non‐permissive temperature of 37°C. Using this assay, we have identified several amino acids in the β1–β2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high‐affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain‐containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non‐permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

Pleckstrin homology domains and the cytoskeleton.

Pleckstrin homology (PH) domains are 100–120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core β‐sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3‐phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3‐kinase activation. Examples of these PH domain‐containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin‐2. PH domain‐mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain‐containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self‐assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.

Epidermal Growth Factor Receptor Dimerization and Activation Require Ligand-Induced Conformational Changes in the Dimer Interface

ABSTRACT Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively “buttressing” the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo- versus heterodimerization.

Golgi localization of glycosyltransferases requires a Vps74p oligomer.

The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. To date, no protein has been identified that recognizes the cytosolic domains of Golgi enzymes and contributes to their localization. Here, we report that yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. We have solved the X-ray crystal structure of Vps74p and find that it forms a tetramer, which we also observe in solution. Deletion of a critical structural motif disrupts tetramer formation and results in loss of Vps74p localization and function. Vps74p is highly homologous to the human GMx33 Golgi matrix proteins, suggesting a conserved function for these proteins in the Golgi enzyme localization machinery.

Matuzumab Binding to EGFR Prevents the Conformational Rearrangement Required for Dimerization

An increasing number of therapeutic antibodies targeting tumors that express the epidermal growth factor receptor (EGFR) are in clinical use or late stages of clinical development. Here we investigate the molecular basis for inhibition of EGFR activation by the therapeutic antibody matuzumab (EMD72000). We describe the X-ray crystal structure of the Fab fragment of matuzumab (Fab72000) in complex with isolated domain III from the extracellular region of EGFR. Fab72000 interacts with an epitope on EGFR that is distinct from the ligand-binding region on domain III and from the cetuximab/Erbitux epitope. Matuzumab blocks ligand-induced receptor activation indirectly by sterically preventing the domain rearrangement and local conformational changes that must occur for high-affinity ligand binding and receptor dimerization.

Extracellular domains drive homo- but not hetero-dimerization of erbB receptors

Many different growth factor ligands, including epidermal growth factor (EGF) and the neuregulins (NRGs), regulate members of the erbB/HER family of receptor tyrosine kinases. These growth factors induce erbB receptor oligomerization, and their biological specificity is thought to be defined by the combination of homo‐ and hetero‐oligomers that they stabilize upon binding. One model proposed for ligand‐induced erbB receptor hetero‐oligomerization involves simple heterodimerization; another suggests that higher order hetero‐oligomers are ‘nucleated’ by ligand‐induced homodimers. To distinguish between these possibilities, we compared the abilities of EGF and NRG1‐β1 to induce homo‐ and hetero‐oligomerization of purified erbB receptor extracellular domains. EGF and NRG1‐β1 induced efficient homo‐oligomerization of the erbB1 and erbB4 extracellular domains, respectively. In contrast, ligand‐induced erbB receptor extracellular domain hetero‐oligomers did not form (except for s‐erbB2–s‐erbB4 hetero‐oligomers). Our findings argue that erbB receptor extracellular domains do not recapitulate most heteromeric interactions of the erbB receptors, yet reproduce their ligand‐induced homo‐oligomerization properties very well. This suggests that mechanisms for homo‐ and hetero‐oligomerization of erbB receptors are different, and contradicts the simple heterodimerization hypothesis prevailing in the literature.