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 Kek1's affinity for EGFR, while class II alleles disrupt Kek1's 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.     

Mechanism of inhibition of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1

The transmembrane protein Kekkon 1 (Kek1) has previously been shown to act in a negative feedback loop to downregulate the Drosophila Epidermal Growth Factor Receptor (DER) during oogenesis. We show that this protein plays a similar role in other DER-mediated developmental processes. Structure-function analysis reveals that the extracellular Leucine-Rich Repeat (LRR) domains of Kek1 are critical for its function through direct association with DER, whereas its cytoplasmic domain is required for apical subcellular localization. In addition, the use of chimeric proteins between Kek1 extracellular and transmembrane domains fused to DER intracellular domain indicates that Kek1 forms an heterodimer with DER in vivo. To characterize more precisely the mechanism underlying the Kek1/DER interaction, we used mammalian ErbB/EGFR cell-based assays. We show that Kek1 is capable of physically interacting with each of the known members of the mammalian ErbB receptor family and that the Kek1/EGFR interaction inhibits growth factor binding, receptor autophosphorylation and Erk1/2 activation in response to EGF. Finally, in vivo experiments show that Kek1 expression potently suppresses the growth of mouse mammary tumor cells derived from aberrant ErbB receptors activation, but does not interfere with the growth of tumor cells derived from activated Ras. Our results underscore the possibility that Kek1 may be used experimentally to inhibit ErbB receptors and point to the possibility that, as yet uncharacterized, mammalian transmembrane LRR proteins might act as modulators of growth factor signalling.     

Comparative analysis of the Kekkon molecules,related members of the LIG superfamily

Leucine-rich repeats (LRRs) and immunoglobulin (Ig) domains represent two of the most abundant sequence elements in metazoan proteomes. Despite this prevalence, comparatively few molecules containing both LRR and Ig (LIG) modules exist, and fewer still have been functionally defined. One LIG whose function has been investigated is the Drosophila protein Kekkon1 (Kek1). In vivo studies have demonstrated a role for Kek1 in Epidermal Growth Factor Receptor (EGFR) signaling and have suggested a role in neuronal pathfinding. Kek1 is the founding member of the Kek family, a group of six Drosophila transmembrane proteins that contain seven LRRs and a single Ig in their extracellular domains. While this arrangement of domains predicts a possible role as cell adhesion molecules (CAMs), to date little is known about the function or evolutionary relationship of these additional Kek molecules. Here we report that orthologs of Kek1, Kek2, Kek5, and Kek6 exist in the mosquito, Anopheles gambiae, and the honeybee, Apis mellifera, indicating that this family has been conserved for ~300 million years of evolutionary time. Comparative sequence analyses reveal remarkable identity among these orthologs, primarily in their extracellular regions. In contrast, the intracellular regions are more divergent, exhibiting only small pockets of conservation. In addition, we provide support for the general notion that these molecules may share common functions as CAMs, by demonstrating that Kek family members can form homotypic and heterotypic complexes.Edited by D. TautzChristina M. MacLaren, Timothy A. Evans and Diego Alvarado contributed equally to this work     

Kekkon5 is an extracellular regulator of BMP signaling

Precise spatial and temporal control of Drosophila Bone Morphogenetic Protein (BMP) signaling is achieved by a host of extracellular factors that modulate ligand distribution and activity. Here we describe Kekkon5 (Kek5), a transmembrane protein containing leucine-rich repeats (LRRs), as a novel regulator of BMP signaling in Drosophila. We find that loss or gain of kek5 disrupts crossvein development and alters the early profile of phosphorylated Mad and dSRF in presumptive crossvein cells. kek5 phenotypic effects closely mimic those observed with Short gastrulation (Sog), but do not completely recapitulate the effects of dominant negative BMP receptors. We further demonstrate that Kek5 is able to antagonize the BMP ligand Glass bottom boat (Gbb) and that the Kek5 LRRs are required for BMP inhibitory activity, while the Ig domain is dispensable in this context. Our identification of Kek5 as a modulator of BMP signaling supports the emerging notion that LIG proteins function as diverse regulators of cellular communication.     

The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis

We have identified the Drosophila transmembrane molecule kekkon 1 (kek1) as an inhibitor of the epidermal growth factor receptor (EGFR) and demonstrate that it acts in a negative feedback loop to modulate the activity of the EGFR tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/EGFR signaling pathway, and loss of kek1 activity is associated with an increase in EGFR signaling. Consistent with our loss-of-function studies, we demonstrate that ectopic overexpression of kek1 mimics a loss of EGFR activity. We show that the extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the EGFR, suggesting potential models for this inhibitory mechanism.     

Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs

Leucine-rich repeat receptor kinases (LRR-RKs) are the largest sub-family of transmembrane receptor kinases in plants. In several LRR-RKs, a loop-out region called an 'island domain', which intercepts the extracellular tandem LRRs at a position near the transmembrane domain, constitutes the ligand-binding pocket, but the absence of the island domain in numerous LRR-RKs raises questions about which domain recognizes the ligand in non-island domain LRR-RKs. Here, we used photoaffinity labeling followed by chemical and enzymatic digestion to show that BAM1, a CLV1/BAM-family LRR-RK whose extracellular domain comprises 22 consecutive LRRs, directly interacts with the small peptide ligand CLE9 at the LRR6-LRR8 region that is relatively distal from the transmembrane domain. Multiple sequence alignment and homology modeling revealed that the inner concave side of LRR6-LRR8 of CLV1/BAM-family LRR-RKs deviates slightly from the LRR consensus. In support of our findings, the clv1-4 mutant carries a missense mutation at the inner concave side of LRR6 of CLV1, and introduction of the corresponding mutation in BAM1 resulted in complete loss of ligand binding activity. Our results indicate that the ligand recognition mechanisms of plant LRR-RKs are more complex and diverse than anticipated.     

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.     

Insulin and epidermal growth factor receptor family members share parallel activation mechanisms

Insulin receptor (IR) and the epidermal growth factor receptor (EGFR) were the first receptor tyrosine kinases (RTKs) to be studied in detail. Both are important clinical targets—in diabetes and cancer, respectively. They have unique extracellular domain compositions among RTKs, but share a common module with two ligand‐binding leucine‐rich‐repeat (LRR)‐like domains connected by a flexible cysteine‐rich (CR) domain (L1‐CR‐L2 in IR/domain, I‐II‐III in EGFR). This module is linked to the transmembrane region by three fibronectin type III domains in IR, and by a second CR in EGFR. Despite sharing this conserved ligand‐binding module, IR and EGFR family members are considered mechanistically distinct—in part because IR is a disulfide‐linked (αβ)₂ dimer regardless of ligand binding, whereas EGFR is a monomer that undergoes ligand‐induced dimerization. Recent cryo‐electron microscopy (cryo‐EM) structures suggest a way of unifying IR and EGFR activation mechanisms and origins of negative cooperativity. In EGFR, ligand engages both LRRs in the ligand‐binding module, “closing” this module to break intramolecular autoinhibitory interactions and expose new dimerization sites for receptor activation. How insulin binds the activated IR was less clear until now. Insulin was known to associate with one LRR (L1), but recent cryo‐EM structures suggest that it also engages the second LRR (albeit indirectly) to “close” the L1‐CR‐L2 module, paralleling EGFR. This transition simultaneously breaks autoinhibitory interactions and creates new receptor‐receptor contacts—remodeling the IR dimer (rather than inducing dimerization per se) to activate it. Here, we develop this view in detail, drawing mechanistic links between IR and EGFR.     

Epidermal growth factor receptor PROTACs as an effective strategy for cancer therapy: A review

Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein that mediates cellular signaling pathways involved in cell proliferation, angiogenesis, apoptosis, and metastatic spread, is an important oncogenic drug target. Targeting the intracellular and extracellular domains of the EGFR has been authorized for a number of small-molecule TKIs and mAbs, respectively. However, their clinical application is limited by EGFR catalytic structural domain alterations, cancer heterogeneity, and persistent drug resistance. To bypass these limitations, protease-targeted chimeras (PROTACs) are emerging as an emerging and promising anti-EGFR therapy. PROTACs compensate for the limitations of traditional occupancy-driven small molecules by exploiting intracellular protein destruction processes. Recently, a mushrooming number of heterobifunctional EGFR PROTACs have been created using wild-type (WT) and mutated EGFR TKIs. PROTACs outperformed EGFR TKIs in terms of cellular inhibition, potency, toxicity profiles, and anti-drug resistance. Herein, we present a comprehensive overview of the development of PROTACs targeting EGFR for cancer therapy, while also highlighting the challenges and opportunities associated with the field.     

Identification of the domain in ErbB2 that restricts ligand-induced degradation

Ligand-induced receptor degradation is an important process for down-regulation of plasma membrane receptors. While epidermal growth factor receptor (EGFR) is rapidly internalised and degraded upon ligand stimulation, ErbB2, the closest member to EGFR in ErbB receptor family, is resistant in ligand-induced degradation. To understand the molecular mechanisms underlying the impairment in ligand-induced degradation of ErbB2, we attempted to determine structural factor in ErbB2 that restricts the degradation. By analysis of ligand-induced degradation of EGFR/ErbB2 chimeras, we have identified a region between amino acid residues F1030 and L1075 in ErbB2 as the domain that restricts the ligand-induced degradation. We designated this domain as the Blocking ErbB2 Degradation or the BED domain. Replacement of the BED domain in an EGFR/ErbB2 chimera with the corresponding region of EGFR changed this chimera from a non-degradable to a degradable receptor, indicating that the BED domain is the factor restricting the ligand-induced degradation of ErbB2. In addition, we found that a non-degradable EGFR/ErbB2 chimera was not defective in tyrosine phosphorylation, ubiquitination and interaction with c-Cbl, rather, was defective in ligand-induced internalisation, suggesting that the endocytosis defect is the cause restricting the degradation of ErbB2, and that c-Cbl-catalysed mono-ubiquitination is not involved in the impairment in ligand-induced degradation of ErbB2.     

Functional role of syndecan-1 cytoplasmic V region in lamellipodial spreading, actin bundling, and cell migration

Cell protrusions contribute to cell motility and migration by mediating the outward extension and initial adhesion of cell edges. In many cells, these extensions are supported by actin bundles assembled by the actin cross-linking protein, fascin. Multiple extracellular cues regulate fascin and here we focus on the mechanism by which the transmembrane proteoglycan, syndecan-1, specifically activates lamellipodial cell spreading and fascin-and-actin bundling when clustered either by thrombospondin-1, laminin, or antibody to the syndecan-1 extracellular domain. There is almost no knowledge of the signaling mechanisms of syndecan-1 cytoplasmic domain and we have tested the hypothesis that the unique V region of syndecan-1 cytoplasmic domain has a crucial role in these processes. By four criteria--the activities of N-cadherin/V region chimeras, syndecan-1 deletion mutants, or syndecan-1 point mutants, and specific inhibition by a membrane-permeable TAT-V peptide--we demonstrate that the V region is necessary and sufficient for these cell behaviors and map the molecular basis for its activity to multiple residues located across the V region. These activities correlate with a V-region-dependent incorporation of cell-surface syndecan-1 into a detergent-insoluble form. We also demonstrate functional roles of syndecan-1 V region in laminin-dependent C2C12 cell adhesion and three-dimensional cell migration. These data identify for the first time specific cell behaviors that depend on signaling through the V region of syndecan-1.     

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.     

A kinase subdomain of transforming growth factor-beta (TGF-beta) type I receptor determines the TGF-beta intracellular signaling specificity.

Transforming growth factor-beta (TGF-beta) signals through a heteromeric complex of related type I and type II serine/threonine kinase receptors. In Mv1Lu cells the type I receptor TbetaRI mediates TGF-beta-induced gene expression and growth inhibition, while the closely related type I receptors Tsk7L and TSR1 are inactive in these responses. Using chimeras between TbetaRI and Tsk7L or TSR1, we have defined the structural requirements for TGF-beta signaling by TbetaRI. The extracellular/transmembrane or cytoplasmic domains of TbetaRI and Tsk7L were functionally not equivalent. The juxtamembrane domain, including the GS motif, and most regions in the kinase domain can functionally substitute for each other, but the alphaC-beta4-beta5 region from kinase subdomains III to V conferred a distinct signaling ability. Replacement of this sequence in TbetaRI by the corresponding domain of Tsk7L inactivated TGF-beta signaling, whereas its introduction into Tsk7L conferred TGF-beta signaling. The differential signaling associated with this region was narrowed down to a sequence of eight amino acids, the L45 loop, which is exposed in the three-dimensional kinase structure and diverges highly between TbetaRI and Tsk7L or TSR1. Replacement of the L45 sequence in Tsk7L with that of TbetaRI conferred TGF-beta responsiveness to the Tsk7L cytoplasmic domain in Mv1Lu cells. Thus, the L45 sequence between kinase subdomains IV and V specifies TGF-beta responsiveness of the type I receptor.     

Antibody-induced dimerization of HARPTPalpha-EGFR chimera suggests a ligand dependent mechanism of regulation for RPTPalpha

We developed a system to study the function of the ectodomain of RPTPalpha, a transmembrane protein-tyrosine phosphatase, by fusing the HA-epitope tagged ectodomain of RPTPalpha to the transmembrane and intracellular domain of the epidermal growth factor receptor, EGFR, a receptor protein-tyrosine kinase that is activated by dimerization. Although the use of chemical crosslinkers shows that preformed HARPTPalpha-EGFR dimers exist, bivalent anti-HA-tag antibody activated HARPTPalpha-EGFR chimeras, suggesting this system may be used to study regulation of dimerization. We used this system to show that newborn calf serum may contain (a) potential ligand(s) for RPTPalpha. Our results suggest that RPTPalpha dimerization and thus activity may be affected by ligand binding.     

Amino acid residues conferring ligand binding properties of prostaglandin I and prostaglandin D receptors. Identification by site-directed mutagenesis

Using chimeras of the mouse prostaglandin (PG) I receptor (mIP) and the mouse PGD receptor (mDP), we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J. Biol. Chem. 272, 15154-15160). In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser(50) in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys(75) and Leu(83) in the second transmembrane domain of mDP confer the high affinity to PGD(2) and the strict specificity of ligand binding of mDP, respectively.     

Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between (835)Ala and (918)Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.     

Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3

The artificial sweetener cyclamate tastes sweet to humans, but not to mice. When expressed in vitro, the human sweet receptor (a heterodimer of two taste receptor subunits: hT1R2 + hT1R3) responds to cyclamate, but the mouse receptor (mT1R2 + mT1R3) does not. Using mixed-species pairings of human and mouse sweet receptor subunits, we determined that responsiveness to cyclamate requires the human form of T1R3. Using chimeras, we determined that it is the transmembrane domain of hT1R3 that is required for the sweet receptor to respond to cyclamate. Using directed mutagenesis, we identified several amino acid residues within the transmembrane domain of T1R3 that determine differential responsiveness to cyclamate of the human versus mouse sweet receptors. Alanine-scanning mutagenesis of residues predicted to line a transmembrane domain binding pocket in hT1R3 identified six residues specifically involved in responsiveness to cyclamate. Using molecular modeling, we docked cyclamate within the transmembrane domain of T1R3. Our model predicts substantial overlap in the hT1R3 binding pockets for the agonist cyclamate and the inverse agonist lactisole. The transmembrane domain of T1R3 is likely to play a critical role in the interconversion of the sweet receptor from the ground state to the active state.