Diego Alvarado

The Juxtamembrane Region of the EGF Receptor Functions as an Activation Domain

In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis and crystallographic approaches, we define a domain within the JM region of the epidermal growth factor receptor (EGFR) that instead plays an activating--rather than autoinhibitory--role. Mutations in the C-terminal 19 residues of the EGFR JM region abolish EGFR activation. In a crystal structure of an asymmetric dimer of the tyrosine kinase domain, the JM region of an acceptor monomer makes extensive contacts with the C lobe of a donor monomer, thus stabilizing the dimer. We describe how an uncharacterized lung cancer mutation in this JM activation domain (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xenograft assay.

ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor

The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular ‘tether’ in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.The orphan receptor tyrosine kinase ErbB2 (HER2/Neu) transforms cells when overexpressed1, and is an important therapeutic target in human cancer2,3. Structural studies4,5 have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular ‘tether’ in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor6. Although ErbB2 is clearly unique among the four human ErbB receptors6,7, we show here that it is the closest structural relative of the single EGF receptor family member (dEGFR) in Drosophila melanogaster. Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands8, yet a crystal structure shows that it too lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches to target novel aspects of this orphan receptor.

Bipartite Inhibition of Drosophila Epidermal Growth Factor Receptor by the Extracellular and Transmembrane Domains of Kekkon1

In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptors activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity we investigated inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1). Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. Here we demonstrate with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. We demonstrate directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, we show that EGFR inhibition is unique to Kek1 among Kek family members and that this function is not ligand or tissue specific. Finally, we have identified a unique class of EGFR alleles that specifically disrupt Kek1 binding and inhibition, but preserve receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, our results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V.

Comparison of Saccharomyces cerevisiae F-BAR Domain Structures Reveals a Conserved Inositol Phosphate Binding Site

F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although they are generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the Saccharomyces cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity and provide a basis for its prediction from sequence.

Ligand regulation of a constitutively dimeric EGF receptor

Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer—resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

Knockouts of Kekkon1 Define Sequence Elements Essential for Drosophila Epidermal Growth Factor Receptor Inhibition

Throughout development, cells utilize feedback inhibition of receptor tyrosine kinase (RTK) signaling as an important means to direct cellular fates. In Drosophila, epidermal growth factor receptor (EGFR) activity is tightly regulated by a complex array of autoregulatory loops, involving an assortment of inhibitory proteins. One inhibitor, the transmembrane protein Kekkon1 (Kek1) functions during oogenesis in a negative feedback loop to directly attenuate EGFR activity. Kek1 contains both leucine-rich repeats (LRRs) and an immunoglobulin (Ig) domain, two of the most prevalent motifs found within metazoan genomes. Here we demonstrate that Kek1 inhibits EGFR activity during eye development and use this role to identify kek1 loss-of-function mutations that implicate the LRRs in directing receptor inhibition. Using a GMR-GAL4, UAS kek1-GFP misexpression phenotype we isolated missense mutations in the kek1 transgene affecting its ability to inhibit EGFR signaling. Genetic, molecular, and biochemical characterization of these alleles indicated that they represent two functionally distinct classes. Class I alleles directly diminish Kek1s affinity for EGFR, while class II alleles disrupt Kek1s subcellular localization, thereby indirectly affecting its ability to associate with and inhibit the receptor. All class I alleles map to the first and second LRRs of Kek1, suggesting a primary role for these two repeats in specifying association with and inhibition of EGFR. Last, our analysis implicates glycine 160 of the second LRR in regulating EGFR binding.

Conservation of an Inhibitor of the Epidermal Growth Factor Receptor, Kekkon1, in Dipterans

Regulation of epidermal growth factor receptor (EGFR) signaling requires the concerted action of both positive and negative factors. While the existence of numerous molecules that stimulate EGFR activity has been well documented, direct biological inhibitors appear to be more limited in number and phylogenetic distribution. Kekkon1 (Kek1) represents one such inhibitor. Kek1 was initially identified in Drosophila melanogaster and appears to be absent from vertebrates and the invertebrate Caenorhabditis. To further investigate Kek1s function and evolution, we identified kek1 orthologs within dipterans. In D. melanogaster, kek1 is a transcriptional target of EGFR signaling during oogenesis, where it acts to attenuate receptor activity through an inhibitory feedback loop. The extracellular and transmembrane portion of Kek1 is sufficient for its inhibitory activity in D. melanogaster. Consistent with conservation of its role in EGFR signaling, interspecies comparisons indicate a high degree of identity throughout these regions. During formation of the dorsal-ventral axis Kek1 is expressed in dorsal follicle cells in a pattern that reflects the profile of receptor activation. D. virilis Kek1 (DvKek1) is also expressed dynamically in the dorsal follicle cells, supporting a conserved role in EGFR signaling. Confirming this, biochemical and transgenic assays indicate that DvKek1 is functionally interchangeable with DmKek1. Strikingly, we find that the cytoplasmic domain contains a region with the highest degree of conservation, which we have implicated in EGFR inhibition and dubbed the Kek tail (KT) box.

Argos Mutants Define an Affinity Threshold for Spitz Inhibition in Vivo

Argos, a secreted antagonist of Drosophila epidermal growth factor receptor (dEGFR) signaling, acts by sequestering the activating ligand Spitz. To understand how different domains in Argos contribute to efficient Spitz sequestration, we performed a genetic screen aimed at uncovering modifiers of an Argos misexpression phenotype in the developing eye. We identified a series of suppressors mapping to the Argos transgene that affect its activity in multiple developmental contexts. These point mutations map to both the N- and C-terminal cysteine-rich regions, implicating both domains in Argos function. We show by surface plasmon resonance that these Argos mutants are deficient in their ability to bind Spitz in vitro. Our data indicate that a mere ∼2-fold decrease in KD is sufficient to compromise Argos activity in vivo. This effect could be recapitulated in a cell-based assay, where a higher molar concentration of mutant Argos was needed to inhibit Spitz-dependent dEGFR phosphorylation. In contrast, a ∼37-fold decrease in the binding constant nearly abolishes Argos activity in vivo and in cellular assays. In agreement with previously reported computational studies, our results define an affinity threshold for optimal Argos inhibition of dEGFR signaling during development.