Vertebrate Lrig3-ErbB Interactions Occur In Vitro but Are Unlikely to Play a Role in Lrig3-Dependent Inner Ear Morphogenesis

Background

The Lrig genes encode a family of transmembrane proteins that have been implicated in tumorigenesis, psoriasis, neural crest development, and complex tissue morphogenesis. Whether these diverse phenotypes reflect a single underlying cellular mechanism is not known. However, Lrig proteins contain evolutionarily conserved ectodomains harboring both leucine-rich repeats and immunoglobulin domains, suggesting an ability to bind to common partners. Previous studies revealed that Lrig1 binds to and inhibits members of the ErbB family of receptor tyrosine kinases by inducing receptor internalization and degradation. In addition, other receptor tyrosine kinase binding partners have been identified for both Lrig1 and Lrig3, leaving open the question of whether defective ErbB signaling is responsible for the observed mouse phenotypes.

Methodology/Principal Findings

Here, we report that Lrig3, like Lrig1, is able to interact with ErbB receptors in vitro. We examined the in vivo significance of these interactions in the inner ear, where Lrig3 controls semicircular canal formation by determining the timing and extent of Netrin1 expression in the otic vesicle epithelium. We find that ErbB2 and ErbB3 are present in the early otic epithelium, and that Lrig3 acts cell-autonomously here, as would be predicted if Lrig3 regulates ErbB2/B3 activity. However, inhibition of ErbB activation in the chick otic vesicle has no detectable effect on Netrin gene expression or canal morphogenesis.

Conclusions/Significance

Our results suggest that although both Lrig1 and Lrig3 can interact with ErbB receptors in vitro, modulation of Neuregulin signaling is unlikely to contribute to Lrig3-dependent processes of inner ear morphogenesis. These results highlight the similar binding properties of Lrig1 and Lrig3 and underscore the need to determine how these two family members bind to and regulate different receptors to affect diverse aspects of cell behavior in vivo.     

Dynamic Analysis of the Epidermal Growth Factor (EGF) Receptor-ErbB2-ErbB3 Protein Network by Luciferase Fragment Complementation Imaging

ErbB3 is a member of the ErbB family of receptor tyrosine kinases. It is unique because it is the only member of the family whose kinase domain is defective. As a result, it is obliged to form heterodimers with other ErbB receptors to signal. In this study, we characterized the interaction of ErbB3 with the EGF receptor and ErbB2 and assessed the effects of Food and Drug Administration-approved therapeutic agents on these interactions. Our findings support the concept that ErbB3 exists in preformed clusters that can be dissociated by NRG-1β and that it interacts with other ErbB receptors in a distinctly hierarchical fashion. Our study also shows that all pairings of the EGF receptor, ErbB2, and ErbB3 form ligand-independent dimers/oligomers. The small-molecule tyrosine kinase inhibitors erlotinib and lapatinib differentially enhance the dimerization of the various ErbB receptor pairings, with the EGFR/ErbB3 heterodimer being particularly sensitive to the effects of erlotinib. The data suggest that the physiological effects of these drugs may involve not only inhibition of tyrosine kinase activity but also a dynamic restructuring of the entire network of receptors.     

Lrig1 is a cell‐intrinsic modulator of hippocampal dendrite complexity and BDNF signaling

Even though many extracellular factors have been identified as promoters of general dendritic growth and branching, little is known about the cell‐intrinsic modulators that allow neurons to sculpt distinctive patterns of dendrite arborization. Here, we identify Lrig1, a nervous system‐enriched LRR protein, as a key physiological regulator of dendrite complexity of hippocampal pyramidal neurons. Lrig1‐deficient mice display morphological changes in proximal dendrite arborization and defects in social interaction. Specifically, knockdown of Lrig1 enhances both primary dendrite formation and proximal dendritic branching of hippocampal neurons, two phenotypes that resemble the effect of BDNF on these neurons. In addition, we show that Lrig1 physically interacts with TrkB and attenuates BDNF signaling. Gain and loss of function assays indicate that Lrig1 restricts BDNF‐induced dendrite morphology. Together, our findings reveal a novel and essential role of Lrig1 in regulating morphogenic events that shape the hippocampal circuits and establish that the assembly of TrkB with Lrig1 represents a key mechanism for understanding how specific neuronal populations expand the repertoire of responses to BDNF during brain development.     

Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling

Maintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- and negative-feedback regulation of instructive extrinsic and intrinsic signals. ErbB signalling is a prerequisite for maintenance of the intestinal epithelium following injury and tumour formation. As ErbB-family ligands and receptors are highly expressed within the stem-cell niche, we hypothesize that strong endogenous regulators must control the pathway in the stem-cell compartment. Here we show that Lrig1, a negative-feedback regulator of the ErbB receptor family, is highly expressed by intestinal stem cells and controls the size of the intestinal stem-cell niche by regulating the amplitude of growth-factor signalling. Intestinal stem-cell maintenance has so far been attributed to a combination of Wnt and Notch activation and Bmpr inhibition. Our findings reveal ErbB activation as a strong inductive signal for stem-cell proliferation. This has implications for our understanding of ErbB signalling in tissue development and maintenance and the progression of malignant disease.     

Lrig1: a new master regulator of epithelial stem cells

Nat Cell Biol 14 4, 401–408 March042012The intestine represents the most vigorously renewing, adult epithelial tissue that makes maintenance of its homeostasis a delicate balance between proliferation, cell cycle arrest, migration, differentiation, and cell death. These processes are precisely controlled by a network of developmental signalling cascades, which include Wnt, Notch, BMP/TGFβ, and Hedgehog pathways. A new, elegant study by Wong et al (2012) now adds Lrig1 as a key player in the control of intestinal homeostasis. As for epidermal stem cells, Lrig1 limits the size of the intestinal progenitor compartment by dampening EGF/ErbB-triggered stem cell expansion.The epithelium of the small intestine is separated into two distinct compartments: a proliferative crypt, containing tissue-specific stem cells, and a villus with differentiated, short-lived cells, which are replenished by a constant stream of cell migration from the underlying crypt (Scoville et al, 2008). In particular, the canonical Wnt pathway in combination with Notch signals control stem cell maintenance and proliferation in the crypt. In addition, both pathways direct differentiation into the Paneth and the absorptive cell lineage, respectively. Intensive cross-talk between the epithelium and the underlying mesenchyme helps to define the crypt–villus boundary. This relies on epithelial-derived Hedgehog and Wnt ligands that trigger stromal BMP production, which in turn signals back to the epithelium to restrict proliferation to the crypt. A gradient of BMP antagonists produced by mesenchymal cells at the bottom of the crypts supports compartmentalization. In addition, a Wnt gradient in the crypt defines EphB expression and establishes repulsion-mediated separation into Paneth cell, proliferative, and differentiation zones along the crypt–villus axis (Figure 1A).Open in a separate windowFigure 1(A) The epithelium of the small intestine contains two populations of multipotent stem cells that reside at the bottom of the crypts. These give rise to transit-amplifying progenitors, which rapidly divide while migrating upwards. Cell cycle arrest and functional differentiation occur when these cells pass from the upper part of the crypt into the villus where they continue their upward movement until they finally undergo apoptosis. Only long-living Paneth cells follow a different path as they migrate downwards to populate the base of the crypt. Control of proliferation and lineage specification of all intestinal epithelial cells is directed in a self-organizing, dynamically regulated process based on cell–cell and cell–environment interactions. Among them, Wnt and Notch signalling have been defined as major determinants for stem cell maintenance, for proliferation of stem cells in the crypt and lineage specification. Epithelial-derived Hedgehog ligands and reciprocal stromal BMP ligands establish a connection between the epithelium and the stroma that regulates the crypt–villus boundary. In addition, repulsive interactions mediated by the Eph/ephrin family allow establishment of stable compartments. Importantly, ErbB signalling, which is partially suppressed by Lrig1 at the base of the crypt, is now shown to be a new key player in the control of stem and progenitor cell expansion. (B) Cross-talk of signalling pathways in intestinal homeostasis with an emphasis on ErbB signalling. A negative feedback loop via Lrig1 helps to fine-tune population size and proliferative activity of intestinal progenitor cells. Lrig1 has been identified as a direct target of Myc and is known to repress ErbB signalling. Myc itself is a main target of the ErbB and Wnt pathways implicated in intestinal stem and progenitor cell expansion. Moreover, Lrig1 has been found to promote BMP signalling, which interferes with intestinal proliferation by restricting AKT activation via PTEN.In the small intestine, two stem cell (SC) populations coexist: Lgr5⁺crypt base columnar cells (CBCs) that cycle every 24 h and are interspersed between Paneth cells, and slower dividing SCs concentrated above (around position +4 relative to the crypt bottom) the Lgr5⁺position (Takeda et al, 2011). The localization of these Hopx⁺mTert⁺slowly cycling SCs partly overlaps with that of quiescent cells, which show long-term label retention upon irradiation damage and pulse labelling with BrdU. Lgr5⁺CBCs are, however, dispensable (Tian et al, 2008) and can be replaced by the second stem cell population, which also shows greater activity during damage repair. The relationship between these two stem cell populations, which can reciprocally generate each other, and the mechanisms that govern quiescence are being elucidated. Importantly, leucine-rich repeats and Ig-like domains 1 (Lrig1), a transmembrane protein that interacts with ErbBs and promotes its degradation, has now been found to be enriched at the crypt base and in the progenitor compartment of the small intestine and colon (Wong et al, 2012). Lrig1 is highly expressed in Lgr5⁺, Musashi1⁺, Ascl2⁺, and Olfm4⁺CBCs, and shows an inverse relation to the pattern of activated, phosphorylated EGFR above the crypt base (Figure 1A). In line with these patterns, deletion of Lrig1 in the mouse causes a dramatic crypt expansion and increased numbers of CBCs, transit-amplifying and Paneth cells. Whether the increase of Paneth cells, which actually do not express Lrig1, is a secondary effect due to the progenitor expansion remains open. Importantly, reduction of EGFR signalling by pharmacological (Gefitinib) and genetic modulation (Egfr^wa-2 mice) is able to partially normalize all Lrig1 phenotypes. These data establish EGF/ErbB signalling, as an important regulator of the crypt compartment, and suggest Lrig1 as a central control that dampens the expansion of stem cells during normal intestinal homeostasis.Lrig1 was initially identified in the skin and proposed to maintain epidermal stem cells in a quiescent state (Watt and Jensen, 2009). Lrig1 marks human interfollicular epidermal stem cells, which can give rise to all epithelial lineages including hair follicle cells in skin reconstitution assays. However, during normal homeostasis, these cells are only bipotent, contributing to the sebaceous gland and the interfollicular epidermis. In contrast to quiescent Lrig1⁺SCs in the skin, Lrig1⁺ intestinal SCs are rapidly dividing and Lrig1 appears to only reduce their proliferative capacity. However, similar to the situation in the skin, Lrig1 and EGF signalling may play an important role during damage repair. Earlier experiments analysed the phenotype of mice lacking major EGF family members (Egger et al, 1997; Troyer et al, 2001). While these mice display some duodenal lesions during normal homeostasis, further experiments established EGF signalling as a key protective component that ameliorates mucosal damage. It remains to be seen whether activation of intestinal SCs during damage repair involves mitigation of Lrig1 dampening.Lrig1 is known to repress ErbB signalling by mediating ubiquitinylation and degradation of activated receptors, thereby limiting the amplitude of EGF signalling (Watt and Jensen, 2009). Consequently, Lrig1 deletion in the intestine induced upregulation of EGFR, ErbB2, and ErbB3, promoting downstream activation of c-Myc within intestinal stem and progenitor cells (Wong et al, 2012). Importantly, Lrig1 is a direct Myc target gene, and thereby part of a negative feedback loop that helps to fine-tune the population size and proliferative activity of intestinal progenitor cells (Figure 1B).Since the rescue of the Lrig1^−/− phenotype by EGFR deficiency was only partial (Wong et al, 2012), other mechanisms may contribute. Intriguingly, Lrig1 has been shown to promote BMP signalling by direct binding to Type I (ALK6) and Type II (ALK1, ALK2, ALK3, and ActRIB) BMP receptors (Gumienny et al, 2010). BMPR1A inactivation, deficiency of its downstream effector PTEN, and transgenic overexpression of the BMP inhibitor Noggin display crypt expansion and increased SC numbers. Inhibition of BMP signalling in these genetic models enhanced AKT activation and increased Wnt signalling, promoting proliferation and adenoma formation (Figure 1B; Scoville et al, 2008). Future work will reveal a potential involvement of BMP and Wnt signalling in the Lrig1 knockout phenotype.The ErbB pathway has been linked to inflammatory bowel disease, and progression and metastatic potential of colorectal cancer. EGFR inhibition blocks adenoma formation in preclinical models, and ErbB pathway inhibition is currently being evaluated in clinical trials with colorectal cancer patients, where promising results have been reported (Cunningham et al, 2004). In contrast, Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumours (Hedman and Henriksson, 2007). Given this heterogeneity, the Lrig1 function in tumours appears to be cell- and context-dependent. Due to early postnatal lethality of Lrig1 knockout mice, the exciting possibility that Lrig1 may act as an intestinal tumour suppressor could not be answered by the current study but clearly deserves further attention.     

SCUBE3 (Signal Peptide-CUB-EGF Domain-containing Protein 3) Modulates Fibroblast Growth Factor Signaling during Fast Muscle Development

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.     

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1^−/−;Lrig2^−/− double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1^−/−;Lrig2^−/− double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member.     

Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.     

Kinase Activator-Receiver Preference in ErbB Heterodimers Is Determined by Intracellular Regions and Is Not Coupled to Extracellular Asymmetry

The EGF receptor (EGFR) family comprises four homologs in humans collectively known as the ErbB or HER proteins. ErbB proteins are receptor tyrosine kinases that become activated when ligands bind to their extracellular regions and promote formation of specific homo- and heterodimers with enhanced tyrosine kinase activity. An essential feature of ErbB activation is formation of an asymmetric kinase dimer in which the C-terminal lobe of one kinase serves as the activator or donor kinase by binding the N-terminal lobe of a receiver or acceptor kinase and stabilizing its active conformation. ErbB extracellular regions are also thought to form active asymmetric dimers in which only one subunit binds ligand. The observation that the unliganded ErbB2 kinase preferentially serves as the activator kinase when paired with EGFR/ErbB1 implied that extracellular asymmetry in ErbB proteins might be coupled to intracellular asymmetry with unliganded partners favoring the activator kinase position. Using cell-based stimulation assays and chimeric ErbB proteins, we show that extracellular asymmetry is not coupled to intracellular asymmetry and that ErbB intracellular regions are sufficient to determine relative kinase activator-receiver orientation. We further show a hierarchy of activator-receiver preferences among ErbB proteins, with EGFR/ErbB1 being the strongest receiver, followed by ErbB2 and then ErbB4, and that cis-phosphorylation of EGFR and ErbB2 appears to be negligible. This hierarchy shapes the nature of signaling responses to different ligands in cells expressing multiple ErbB proteins.     

Stretch-induced fetal type II cell differentiation is mediated via ErbB1-ErbB4 interactions

Stretch-induced differentiation of lung fetal type II epithelial cells is mediated through EGFR (ErbB1) via release of HB-EGF and TGF-α ligands. Employing an EGFR knock-out mice model, we further investigated the role of the ErbB family of receptors in mechanotranduction during lung development. Deletion of EGFR prevented endogenous and mechanical stretch-induced type II cell differentiation via the ERK pathway, which was rescued by overexpression of a constitutively active MEK. Interestingly, the expression of ErbB4, the only ErbB receptor that EGFR co-precipitates in wild-type cells, was decreased in EGFR-deficient type II cells. Similar to EGFR, ErbB4 was activated by stretch and participated in ERK phosphorylation and type II cell differentiation. However, neuregulin (NRG) or stretch-induced ErbB4 activation were blunted in EGFR-deficient cells and not rescued after ErbB4 overexpression, suggesting that induction of ErbB4 phosphorylation is EGFR-dependent. Finally, we addressed how shedding of ligands is regulated by EGFR. In knock-out cells, TGF-α, a ligand for EGFR, was not released by stretch, while HB-EGF, a ligand for EGFR and ErbB4, was shed by stretch although to a lower magnitude than in normal cells. Release of these ligands was inhibited by blocking EGFR and ERK pathway. In conclusion, our studies show that EGFR and ErbB4 regulate stretch-induced type II cell differentiation via ERK pathway. Interactions between these two receptors are important for mechanical signals in lung fetal type II cells. These studies provide novel insights into the cell signaling mechanisms regulating ErbB family receptors in lung cell differentiation.     

The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.     

Lrig3 regulates neural crest formation in Xenopus by modulating Fgf and Wnt signaling pathways

Leucine-rich repeats and immunoglobulin-like domains 3 (Lrig3) was identified by microarray analysis among genes that show differential expression during gastrulation in Xenopus laevis. Lrig3 was expressed in the neural plate and neural crest (NC) at neurula stages, and in NC derivatives and other dorsal structures during tailbud stages. A prominent consequence of the morpholino-induced inhibition of Lrig3 expression was impaired NC formation, as revealed by the suppression of marker genes, including Slug, Sox9 and Foxd3. In the NC induction assay involving Chordin plus Wnt3a-injected animal caps, Lrig3 morpholino inhibited expression of Slug, Sox9 and Foxd3, but not of Pax3 and Zic1. In line with this, Lrig3 knockdown prevented NC marker induction by Pax3 and Zic1, suggesting that Lrig3 acts downstream of these two genes in NC formation. Injection of Lrig3 and Wnt3a led to low-level induction of NC markers and enhanced induction of Fgf3, Fgf4 and Fgf8 in animal caps, suggesting a positive role for Lrig3 in Wnt signaling. Lrig3 could attenuate Fgf signaling in animal caps, did interact with Fgf receptor 1 in cultured cells and, according to context, decreased or increased the induction of NC markers by Fgf. We suggest that Lrig3 functions in NC formation in Xenopus by modulating the Wnt and Fgf signaling pathways.     

Plasticity in interactions of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlies promiscuity of FGF1

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of fibroblast growth factor receptors 1–3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived fibroblast growth factors (FGFs), respectively. FGF1 is termed the “universal FGFR ligand” because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the “b” and “c” splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-19, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.     

Destabilization of the Epidermal Growth Factor Receptor (EGFR) by a Peptide That Inhibits EGFR Binding to Heat Shock Protein 90 and Receptor Dimerization

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.     

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers.