Mark A. Lemmon

Cell Signaling by Receptor Tyrosine Kinases

Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses.

Membrane recognition by phospholipid-binding domains

Many different globular domains bind to the surfaces of cellular membranes, or to specific phospholipid components in these membranes, and this binding is often tightly regulated. Examples include pleckstrin homology and C2 domains, which are among the largest domain families in the human proteome. Crystal structures, binding studies and analyses of subcellular localization have provided much insight into how members of this diverse group of domains bind to membranes, what features they recognize and how binding is controlled. A full appreciation of these processes is crucial for understanding how protein localization and membrane topography and trafficking are regulated in cells.

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.

Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation

Heparin is required for fibroblast growth factor (FGF) stimulation of biological responses. Using isothermal titration calorimetry, we show that acidic FGF (aFGF) forms a 1:1 complex with the soluble extracellular domain of FGF receptor (FGFR). Heparin exerts its effect by binding to many molecules of aFGF. The resulting aFGF-heparin complex can bind to several receptor molecules, leading to FGFR dimerization. In two cell lines lacking endogenous heparan sulfate, exogenous heparin is required for FGFR dimerization, tyrosine kinase activation, c-fos mRNA transcription, and cell proliferation. Moreover, a synthetic heparin analog that binds monovalently to aFGF blocks FGFR dimerization, activation, and signaling via FGFR. We propose that heparin causes oligomerization of aFGF such that its binding to FGFR results in dimerization and activation. This represents a novel mechanism for transmembrane signaling and may account for the action of many heparin-bound growth factors.

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.

Activation of phospholipase Cγ by PI 3‐kinase‐induced PH domain‐mediated membrane targeting

Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase Cγ (PLCγ) and phosphatidylinositol (PI) 3‐kinase. While it is well established that tyrosine phosphorylation of PLCγ is necessary for its activation, we show here that PLCγ is regulated additionally by the lipid products of PI 3‐kinase. We demonstrate that the pleckstrin homology (PH) domain of PLCγ binds to phosphatidylinositol 3,4,5‐trisphosphate [PdtIns(3,4,5)P3], and is targeted to the membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not membrane targeted. Consistent with these observations, activation of PI 3‐kinase causes PLCγ PH domain‐mediated membrane targeting and PLCγ activation. By contrast, either the inhibition of PI 3‐kinase by overexpression of a dominant‐negative mutant or the prevention of PLCγ membrane targeting by overexpression of the PLCγ PH domain prevents growth factor‐induced PLCγ activation. These experiments reveal a novel mechanism for cross‐talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.

Phosphoinositide Recognition Domains

Domains or modules known to bind phosphoinositides have increased dramatically in number over the past few years, and are found in proteins involved in intracellular trafficking, cellular signaling, and cytoskeletal remodeling. Analysis of lipid binding by these domains and its structural basis has provided significant insight into the mechanism of membrane recruitment by the different cellular phosphoinositides. Domains that target only the rare (3‐phosphorylated) phosphoinositides must bind with very high affinity, and with exquisite specificity. This is achieved solely by headgroup interactions in the case of certain pleckstrin homology (PH) domains [which bind PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2], but requires an additional membrane‐insertion and/or oligomerization component in the case of the PtdIns(3)P‐targeting phox homology (PX) and FYVE domains. Domains that target PtdIns(4,5)P2, which is more abundant by some 25‐fold, do not require the same stringent affinity and specificity characteristics, and tend to be more diverse in structure. The mode of phosphoinositide binding by different domains also appears to reflect their distinct functions. For example, pleckstrin homology domains that serve as simple targeting domains recognize only phosphoinositide headgroups. By contrast, certain other domains, notably the epsin ENTH domain, appear to promote bilayer curvature by inserting into the membrane upon binding.

Regulation of signal transduction and signal diversity by receptor oligomerization.

Receptor oligomerization was initially proposed as a mechanism by which epidermal growth factor activates the protein tyrosine kinase activity of its receptor. It is now well established that ligand-induced receptor oligomerization plays an important role in transmembrane signaling by a large number of receptors for hormones, cytokines and growth factors. Heterodimerization of the extracellular domains of two members of the same receptor family, or interaction with an accessory molecule, can increase the diversity of ligands recognized by individual receptors. Heterodimerization of cytoplasmic domains permits the recruitment of different complements of SH2-domain-containing signaling molecules, increasing the repertoire of signaling pathways that can be activated by a given receptor.

Regulation of growth factor activation by proteoglycans: What is the role of the low affinity receptors?

Department of Pharmacology New York University Medical Center New York, New York 10016 Many cell membrane receptors, such as the nicotinic ace- tylcholine receptor and the T cell antigen receptor, are composed of several different subunits, and their correct assembly is necessary for the generation of functional re- ceptors. Most lymphokine receptors are composed of at least two components, and ligand-induced oligomeriza- tion is essential for receptor activation and signal transmis- sion. In recent years, it has become clear that various growth factors and lymphokines can bind to two different classes of cell surface receptors. For example, fibroblast growth factor (FGF) and transforming growth factor p (TGFB) both bind with high affinity to signaling receptors endowed with tyrosine or serinelthreonine kinase activi- ties. However, the same growth factors also bind with lower affinity to cell surface proteoglycans that cannot transmit signals alone, but somehow modulate the ability of the growth factor or the signaling receptor to generate a biological response (Klagsbrun and Baird, 1991; L6pez- Casillas et al., 1993; Yayon et al., 1991; Roghani et al., 1994). Proteoglycans are proteins that are found predomi- nantly on the cell surface and in the extracellular matrix and that contain carbohydrates called glycosaminogly- cans. Glycosaminoglycans are polymers of disaccharide repeats, which are mostly highly sulfated and negatively charged. The main glycosaminoglycans in proteoglycans are chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate (Ruoslahti, 1989). Binding of growth factors to proteoglycans is thought to have an important regulatory role (Ruoslahti and Yamaguchi, 1991). This has been particularly well explored for FGF, in which it has been shown that heparins (or heparan sulfate proteoglycans) are necessary for FGF-induced biologicl neurotrophin binding to ~75 (low affinity) and to the various nerve growth factor (NGF) receptor tyrosine kinases (trkA, trkB, and trkC, which are high affinity), as well as the binding of the insulin- like growth factor IGF2 to the IGF2 receptor (nonsignaling receptor) and to the IGFl receptor (a tyrosine kinase related to the insulin receptor). The existence of nonsignaling as well as signaling receptors for the same ligand is a feature common for many growth factors. How- ever, the physiological role of the nonsignaling receptors is poorly understood. The most discussed model for the role of the low affinity nonsignaling receptors is that they present ligand to high affinity signaling receptors. Binding of the ligand to the high affinity signaling receptors will then activate this receptor and trigger biological responses. However, when a given ligand at low concentration is allowed to bind to cells that display on their surface both low and high affinity receptors, the law of mass action dictates that at equilib- rium the ligand will bind, predominantly, to high affinity receptors rather than to the low affinity receptors. This fact makes it difficult to accept the so-called presentation model. At higher ligand concentration, both the high and low affinity receptors will be occupied. The binding con- stants of the high affinity receptors are usually lo- to 1 OO- fold higher than those of the low affinity receptors. There- fore, even when the density of the low affinity receptors on the cell surface is higher by an order of magnitude, a similar number of high and low affinity receptors will be occupied upon saturation of the high affinity receptors. These arguments demonstrate the need for a different model to understand the role of the low affinity receptors. An alternative and more plausible model is that the primary function of these receptors is to reduce the dimensionality of ligand diffusion from three to two dimensions (Adam and Delbruck, 1968; Richter and Eigen, 1974). When ligands such as lymphokines or growth factors are bound to cell surface receptors, their diffusion is restricted to two dimen- sions ratherthan diffusing in thethree-dimensionalvolume of the extracellular space. Once restricted to just two di- mensions, the ligand molecules are more likely to encoun- ter and bind to the less abundant high affinity signaling receptors. In otherwords, binding of ligands, such as FGF, to abundant low affinity receptors that are restricted two dimensions will increase the local concentration of the bound ligands at the plasma membrane, and the probabil- ity of their interaction with a high affinity receptor will be greatly enhanced. For example, 20,000 receptors per cell