Intracellular Juxtamembrane Region Research Articles

Molecular mechanism of IgE-mediated FcεRI activation.

Allergic diseases, affecting over a quarter of individuals in industrialized countries, have become significant public health concerns1,2. The high-affinity Fc receptor for IgE (FcεRI), mainly present on mast cells and basophils, plays a crucial role in allergic diseases3-5. Monomeric IgE binding to FcεRI regulates mast cell survival, differentiation, and maturation6-8. However, the underlying molecular mechanism remains unclear. Here we demonstrate that, prior to IgE binding, FcεRI mostly exists as a homo-dimer on human mast cell membrane. The structure of human FcεRI confirms the dimeric organization, with each promoter comprising one α subunit, one β subunit, and two γ subunits. The transmembrane helices of the α subunits form a layered arrangement with those of the γ and β subunits. The dimeric interface is mediated by a four-helix bundle of the α and γ subunits at the intracellular juxtamembrane region. Cholesterol-like molecules embedded within the transmembrane domain may stabilize the dimeric assembly. Upon IgE binding, the dimeric FcεRI dissociates into two protomers, each binding to an IgE molecule. Importantly, this process elicits transcriptional activation of Egr1/3 and Ccl2 in rat basophils, which can be attenuated by inhibiting the FcεRI dimer-to-monomer transition. Collectively, our study unveils the mechanism of antigen-independent, IgE-mediated FcεRI activation.

PDGF-induced internalisation promotes proteolytic cleavage of PDGFRβ in mesenchymal cells

Platelet-derived growth factor (PDGF)-induced signalling via PDGF receptor β (PDGFRβ) leads to activation of downstream signalling pathways which regulate multiple cellular responses. It is unclear how PDGFRβ is degraded; both lysosomal and proteasomal degradation have been suggested. In this study, we have characterised the proteolytic cleavage of ligand-activated PDGFRβ, which results in two fragments: a larger fragment containing the extracellular domain, the transmembrane segment, and a part of the intracellular juxtamembrane region with a molecular mass of ∼130 kDa, and an intracellular ∼70 kDa fragment released into the cytoplasm. The proteolytic processing did not take place without internalisation of PDGFRβ. In addition, chelation of intracellular Ca2+ inhibited proteolytic processing. Inhibition of the proteasome affected signal transduction by increasing the phosphorylation of PDGFRβ, PLCγ, and STAT3 while reducing it on Erk1/2 and not affecting Akt. The proteolytic cleavage was observed in fibroblasts or cells that had undergone epithelial-mesenchymal transition.

Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling.

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

Two-step release of kinase autoinhibition in discoidin domain receptor 1

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase with important functions in organogenesis and tissue homeostasis. Aberrant DDR1 activity contributes to the progression of human diseases, including fibrosis and cancer. How DDR1 activity is regulated is poorly understood. We investigated the function of the long intracellular juxtamembrane (JM) region of human DDR1 and found that the kinase-proximal segment, JM4, is an important regulator of kinase activity. Crystal structure analysis revealed that JM4 forms a hairpin that penetrates the kinase active site, reinforcing autoinhibition by the activation loop. Using in vitro enzymology with soluble kinase constructs, we established that release from autoinhibition occurs in two distinct steps: rapid autophosphorylation of the JM4 tyrosines, Tyr569 and Tyr586, followed by slower autophosphorylation of activation loop tyrosines. Mutation of JM4 tyrosines abolished collagen-induced DDR1 activation in cells. The insights may be used to develop allosteric, DDR1-specific, kinase inhibitors.

Clustering, Spatial Distribution, and Phosphorylation of Discoidin Domain Receptors 1 and 2 in Response to Soluble Collagen I

Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that signal in response to collagen. We had previously shown that collagen binding leads to clustering of DDR1b, a process partly mediated by its extracellular domain (ECD). In this study, we investigated (i) the impact of the oligomeric state of DDR2 ECD on collagen binding and fibrillogenesis, (ii) the effect of collagen binding on DDR2 clustering, and (iii) the spatial distribution and phosphorylation status of DDR1b and DDR2 after collagen stimulation. Studies were conducted using purified recombinant DDR2 ECD proteins in monomeric, dimeric or oligomeric state, and MC3T3-E1 cells expressing full-length DDR2-GFP or DDR1b-YFP. We show that the oligomeric form of DDR2 ECD displayed enhanced binding to collagen and inhibition of fibrillogenesis. Using atomic force and fluorescence microscopy, we demonstrate that unlike DDR1b, DDR2 ECD and DDR2-GFP do not undergo collagen-induced receptor clustering. However, after prolonged collagen stimulation, both DDR1b-YFP and DDR2-GFP formed filamentous structures consistent with spatial re-distribution of DDRs in cells. Immunocytochemistry revealed that while DDR1b clusters co-localized with non-fibrillar collagen, DDR1b/DDR2 filamentous structures associated with collagen fibrils. Antibodies against a tyrosine phosphorylation site in the intracellular juxtamembrane region of DDR1b displayed positive signals in both DDR1b clusters and filamentous structures. However, only the filamentous structures of both DDR1b and DDR2 co-localized with antibodies directed against tyrosine phosphorylation sites within the receptor kinase domain. Our results uncover key differences and similarities in the clustering abilities and spatial distribution of DDR1b and DDR2 and their impact on receptor phosphorylation.

Cell-Surface Expression Levels Are Important for Fine-Tuning the Performance of Receptor Tyrosine Kinase-Based Signalobodies.

As receptor tyrosine kinases (RTKs) play important roles in cell-fate control of various cell types, engineered RTKs that could respond to inexpensive ligands might drastically reduce the cost of producing desired cells for various applications in regenerative medicine. We developed several engineered RTKs named "signalobodies" in which the ligand-recognition domain of RTKs is replaced by single-chain Fv for enabling recognition of a specific antigen. However, the remaining concern was the dysregulation of antigen-dependent on/off signaling of the signalobodies. This study aims at fine-tuning the performance of the signalobodies based on three RTKs (fibroblast growth factor receptor 1, insulin receptor, and c-fms). To this end, the cell-surface expression levels of the RTK-based signalobodies were altered by locating their genes either upstream or downstream of the internal ribosomal entry site, and by inserting 1 to 3 alanine residue(s) at the intracellular juxtamembrane region. As a result, while the signaling response was different among the three signalobodies, the antigen-dependent on/off regulation became tighter when the cell-surface expression levels of the signalobodies were lowered. Therefore, we successfully developed a method to diminish the leaky signaling of RTK-based signalobodies, which will be important for establishing the signalobody-based platform technology that can produce cells of interest for regenerative medicine.

Domain-dependent effects of insulin and IGF-1 receptors on signalling and gene expression

Despite a high degree of homology, insulin receptor (IR) and IGF-1 receptor (IGF1R) mediate distinct cellular and physiological functions. Here, we demonstrate how domain differences between IR and IGF1R contribute to the distinct functions of these receptors using chimeric and site-mutated receptors. Receptors with the intracellular domain of IGF1R show increased activation of Shc and Gab-1 and more potent regulation of genes involved in proliferation, corresponding to their higher mitogenic activity. Conversely, receptors with the intracellular domain of IR display higher IRS-1 phosphorylation, stronger regulation of genes in metabolic pathways and more dramatic glycolytic responses to hormonal stimulation. Strikingly, replacement of leucine973 in the juxtamembrane region of IR to phenylalanine, which is present in IGF1R, mimics many of these signalling and gene expression responses. Overall, we show that the distinct activities of the closely related IR and IGF1R are mediated by their intracellular juxtamembrane region and substrate binding to this region.

Promiscuous signaling of ligands via mutant ALK2 in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary disorder characterized by successive heterotopic bone formation, for which at present there is no therapy. Mutations in the bone morphogenetic protein (BMP) type I receptor Activin receptor-like kinase 2 (ACVR1/ALK2) are the main trigger for FOP and inflammation is thought to be the secondary hit. The single nucleotide mutation at position 617 in the cDNA ALK2 sequence, which is found in 98% of FOP patients, results in a R206H change in the intracellular juxtamembrane region of ALK2. Previous studies had revealed that this mutation perturbs the interaction with the negative regulator FKBP12, thereby sensitising cells expressing this mutant receptor to BMPs, which are potent inducers of cartilage and bone formation. Recently, however, a twist in the underlying mechanism of FOP was revealed. Mutant ALK2 was found to respond to Activin-A, whereas wild type ALK2 function is inhibited by Activin-A. The latter cytokine is induced locally upon tissue damage and inflammation. Moreover, therapeutic targeting of Activin-A was found to inhibit heterotopic ossification in a mutant ALK2 knock-in mouse model that is highly reminiscent to human FOP. This review will focus on these latest surprising findings and discuss the implication for treatment of FOP patients.

Antitumor Properties of an IgG2-Enhanced Next-Generation MET Monoclonal Antibody That Degrades Wild-Type and Mutant MET Receptors.

A sound rationale exists for antibody targeting of the MET receptor tyrosine kinase, but therapeutic agents that can broadly block HGF ligand binding and exon 14-mutated or amplified MET to induce receptor degradation have yet to be reported. Here we report the identification of several MET monoclonal antibodies (mAb) that block MET-dependent signaling and tumor growth. In particular, the MET mAb KTN0073 and KTN0074 bind the Sema/PSI domain, at overlapping but distinct epitopes, preventing HGF interaction with MET and triggering receptor ubiquitination and degradation. Notably, both mAbs also triggered degradation of oncogenic MET exon 14 mutants, which propagate more durable MET signals due to a defect in receptor degradation. Mechanistic investigations showed that both mAbs engaged a pathway distinct from HGF-induced receptor degradation and protease-mediated shedding, independently of signaling driven by the exon 14-encoded sequences in the intracellular juxtamembrane region of the MET receptor. Grafting the mAb variable regions onto the IgG2 constant region dramatically enhanced the tumor inhibitory activities of KTN0073 but not KTN0074, suggesting a specific influence of antibody isotype of the epitopes for these two MET mAbs. Overall, our results highlight KTN0073 as a novel IgG2-based MET mAb that acts through exon 14-independent mechanisms to degrade the MET receptor, potentially offering a therapeutic tool to treat a broader range of human tumors where MET is exon 14 mutated or amplified. Cancer Res; 76(19); 5788-97. ©2016 AACR.

Abstract 3835: An anti-MET IgG2 monoclonal antibody degrades both wild-type and exon 14 mutant MET receptor tyrosine kinase through a novel exon 14-independent mechanism and inhibits tumor growth

Abstract MET gene amplification and exon 14 mutations have been reported and implicated as oncogenic drivers in several types of cancer representing approximately 5-7% of NSCLC, 4-10% of gastric cancer, and 5-10% of colon cancers. Preclinical research and clinical data derived from patients with tumors containing MET gene amplification and exon 14 mutations showed significant growth inhibition and meaningful clinical benefit after treatment with MET-targeting tyrosine kinase inhibitors (TKIs), supporting the hypothesis that MET gene amplification and exon 14 mutations are actionable biomarkers. We have identified several anti-MET monoclonal antibodies that block MET-dependent signaling and tumor growth through: (1) binding to the Sema/PSI domain, (2) preventing HGF interaction with MET and (3) inducing receptor ubiqutination and degradation. One of the anti-MET antibodies, KTN0073, induces potent degradation of oncogenic exon 14 mutant MET, which has been reported to propagate prolonged MET signaling due to a defect in receptor degradation. Our data suggest that KTN0073 engages a pathway different from HGF-induced receptor degradation and protease-mediated shedding, which is independent of the 47 amino acids that are encoded by exon 14 in the intracellular juxtamembrane region and only partially inhibited by the TKI crizotinib. In summary, we report the use of an anti-MET monoclonal antibody to elucidate an exon 14-independent mechanism of MET degradation that could provide a novel way to design anti-MET therapeutics to inhibit growth of human tumors that are driven by HGF, MET amplification and exon 14 mutations. Based on this work, KTN0073-IgG2 anti-MET antibody has been humanized for further preclinical development to explore its potential as a therapeutic for the treatment of human MET expressing cancers. Citation Format: Brett S. Robinson, Sreekala Mandiyan, Gerald McMahon, Yan Yang. An anti-MET IgG2 monoclonal antibody degrades both wild-type and exon 14 mutant MET receptor tyrosine kinase through a novel exon 14-independent mechanism and inhibits tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3835.

Leda-1/Pianp is targeted to the basolateral plasma membrane by a distinct intracellular juxtamembrane region and modulates barrier properties and E-Cadherin processing

Leda-1/Pianp is a type-I transmembrane protein which is sorted to the basolateral membrane domain of polarized epithelial cells. Here, we investigated trafficking mechanisms and functions of Leda-1/Pianp in MDCK and MCF-7 cells. Basolateral sorting and posttranslational modifications depended on the intracellular juxtamembrane region. Functionally, Leda-1/Pianp increased the transepithelial electrical resistance generated by a polarized cell sheet. Furthermore, resistance to junctional destabilization by tumor cells was enhanced by Leda-1/Pianp indicating increased stability and tightness of intercellular junctions. While Claudin 1 and 4 expression and activities of small GTPases were not affected, γ-Secretase-mediated cleavage of E-Cadherin was attenuated by Leda-1/Pianp. Regulation of proteolytic processing is thus a molecular mechanism by which Leda-1/Pianp can affect junctional integrity and function.

Mutual cross-talk between fibronectin integrins and the EGF receptor

Extension of the plasma membrane is one of the first steps in cell migration. Understanding how cells “choose” between various types of membrane protrusion enhances our knowledge of both normal and cancer cell physiology. The EGF receptor is a paradigm for understanding how transmembrane receptor tyrosine kinases regulate intracellular signaling following ligand stimulation. Evidence from the past decade indicates that EGF receptors also form macromolecular complexes with integrin receptors leading to EGF receptor transactivation during cell adhesion. However, relatively little is known about how these complexes form and impact cell migration. Our recent work characterized a molecular complex between EGF receptor and β3 integrin which recognizes RGD motifs in extracellular matrix proteins. Complex formation requires a dileucine motif (679-LL) in the intracellular juxtamembrane region of the EGF receptor that also controls whether or not the receptor undergoes Src kinase-dependent phosphorylation at Tyr-845. In contrast to wild-type receptors, mutant EGF receptors defective for Tyr-845 phosphorylation form complexes with β1 integrin that also binds RGD motifs. In addition, we have discovered that EGF receptor antagonizes small GTPase RhoA by mediating membrane recruitment of its regulatory GAP p190RhoGAP. In this addendum we discuss a potential new role for Src-dependent EGF receptor transactivation in integrin/EGF receptor complex formation. We also discuss how our study fits with previous observations linking p190RhoGAP to RhoA-dependent cytoskeletal rearrangements involved in cell migration, and provide new data that the EGF receptor is compartmentalized to relatively immature zyxin-poor focal adhesions which are the likely site of p190RhoGAP signaling.

KCNE4 Juxtamembrane Region Is Required for Interaction with Calmodulin and for Functional Suppression of KCNQ1

Voltage-gated potassium (K(V)) channels, such as KCNQ1 (K(V)7.1), are modulated by accessory subunits and regulated by intracellular second messengers. Accessory subunits belonging to the KCNE family exert diverse functional effects on KCNQ1, have been implicated in the pathogenesis of various genetic disorders of heart rhythm, and contribute to transducing intracellular signaling events into changes in K(V) channel activity. We investigated the interactions between calmodulin (CaM), the ubiquitous Ca(2+)-transducing protein that binds and confers Ca(2+) sensitivity to the biophysical properties of KCNQ1, and KCNE4. These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel. We determined that KCNE4, but not KCNE1, can biochemically interact with CaM and that this interaction is Ca(2+)-dependent and requires a tetraleucine motif in the juxtamembrane region of the KCNE4 C terminus. Furthermore, disruption of the KCNE4-CaM interaction either by mutagenesis of the tetraleucine motif or by acute Ca(2+) chelation impairs the ability of KCNE4 to inhibit KCNQ1. Our findings have potential relevance to KCNQ1 regulation both by KCNE accessory subunits and by an important intracellular signaling molecule.

Four novel RET germline variants in exons 8 and 11 display an oncogenic potential in vitro

Most germline-activating mutations of the RET proto-oncogene associated with inherited medullary thyroid cancer (MTC) are localized in exons 10, 11 and 13-15. Four novel RET variants, located in the extracellular domain (p.A510V, p.E511K and p.C531R) coded by exon 8 and in the intracellular juxtamembrane region (p.K666N) coded by exon 11, were identified on the leukocyte DNA from apparently sporadic cases. Plasmids carrying Ret9-wild-type (Ret9-WT), Ret9-C634R and all Ret9 variants were transfected, and the phosphorylation levels of RET and ERK were evaluated by western blot analyses. The transforming potentials were assessed by the focus formation assay. The p.A510V, p.E511K and p.C531R variants were found to generate RET and ERK phosphorylation levels and to have a transforming activity higher than that of Ret9-WT variant, but lower than that of Ret9-C634R variant. Differently, the p.K666N variant, located immediately downstream of the transmembrane domain, and involving a conserved residue, displayed high kinase and transforming activities. Computational analysis predicted non-conservative alterations in the mutant proteins consistent with putative modifications of the receptor conformation. The molecular analyses revealed an oncogenic potential for all the novel germline RET variants. Therefore, the prevalence of exon 8 genomic variations with an oncogenic potential may be higher than previously thought, and the analysis of this exon should be considered after the exclusion of mutations in the classical hotspots. In addition, on the basis of these functional data, it is advisable to extend the genetic screening to all the first-degree relatives of the MTC patients, and to perform a strict follow-up of familial carriers.

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.

The Influence of Domain Structures on the Signal Transduction of Chimeric Receptors Derived from the Erythropoietin Receptor

Although cytokine receptors regulate many cellular functions, contribution of receptor's domains and their conformation to signal transduction remains unclear. In this study, we designed a series of chimeric erythropoietin receptor (EpoR) variants encoding a haemagglutinin epitope-tagged anti-fluorescein single-chain Fv and different combinations of extracellular D1/D2 domain(s) of EpoR as the extracellular domain to allow the receptor to be activated by multiple ligands. Furthermore, one to four Ala residues were inserted at the intracellular juxtamembrane region of each chimeric receptor to modulate the conformation of the intracellular domain. When the chimeric receptors were expressed in Ba/F3 cells, cell-surface expression levels of chimeric receptors without D2 domain were markedly lowered, suggesting a role of D2 domain for stabilizing the receptor. Furthermore, the ligand-dependent cell proliferation was strongly affected by extracellular domain structures and the number of inserted Ala residues. Moreover, the conformational change of chimeric receptors was induced by various ligands to detect the phosphorylation of JAK2, STAT5 and ERK2, whose activations are characteristics of EpoR signalling. Consequently, the phosphorylation pattern of these signal transducers was significantly influenced by ligands and receptor variants. These results indicate that signal transduction of EpoR is strongly affected by conformation of both extracellular and intracellular domains.

Epidermal growth factor receptor juxtamembrane region regulates allosteric tyrosine kinase activation

Structural studies of the extracellular and tyrosine kinase domains of the epidermal growth factor receptor (ErbB-1) provide considerable insight into facets of the receptor activation mechanism, but the contributions of other regions of ErbB-1 have not been ascertained. This study demonstrates that the intracellular juxtamembrane (JM) region plays a vital role in the kinase activation mechanism. In the experiments described herein, the entire ErbB-1 intracellular domain (ICD) has been expressed in mammalian cells to explore the significance of the JM region in kinase activity. Deletion of the JM region (DeltaJM) results in a severe loss of ICD tyrosine phosphorylation, indicating that this region is required for maximal activity of the tyrosine kinase domain. Coexpression of DeltaJM and dimerization-deficient kinase domain ICD mutants revealed that the JM region is indispensable for allosteric kinase activation and productive monomer interactions within a dimer. Studies with the intact receptor confirmed the role of the JM region in kinase activation. Within the JM region, Thr-654 is a known protein kinase C (PKC) phosphorylation site that modulates kinase activity in the context of the intact ErbB-1 receptor; yet, the mechanism is not known. Whereas a T654A mutation promotes increased ICD tyrosine phosphorylation, the phosphomimetic T654D mutant generates a 50% reduction in ICD tyrosine phosphorylation. Similar to the DeltaJM mutants, the T654D mutant ICD failed to interact with a wild-type monomer. This study reveals an integral role for the intracellular JM region of ErbB-1 in allosteric kinase activation.

Cdk5 Is Involved in BDNF-Stimulated Dendritic Growth in Hippocampal Neurons

Neurotrophins are key regulators of neuronal survival and differentiation during development. Activation of their cognate receptors, Trk receptors, a family of receptor tyrosine kinases (RTKs), is pivotal for mediating the downstream functions of neurotrophins. Recent studies reveal that cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase, may modulate RTK signaling through phosphorylation of the receptor. Given the abundant expression of both Cdk5 and Trk receptors in the nervous system, and their mutual involvement in the regulation of neuronal architecture and synaptic functions, it is of interest to investigate if Cdk5 may also modulate Trk signaling. In the current study, we report the identification of TrkB as a Cdk5 substrate. Cdk5 phosphorylates TrkB at Ser478 at the intracellular juxtamembrane region of TrkB. Interestingly, attenuation of Cdk5 activity or overexpression of a TrkB mutant lacking the Cdk5 phosphorylation site essentially abolishes brain-derived neurotrophic factor (BDNF)–triggered dendritic growth in primary hippocampal neurons. In addition, we found that Cdk5 is involved in BDNF-induced activation of Rho GTPase Cdc42, which is essential for BDNF-triggered dendritic growth. Our observations therefore reveal an unanticipated role of Cdk5 in TrkB-mediated regulation of dendritic growth through modulation of BDNF-induced Cdc42 activation.

N-methyl-d-glucamine and propidium dyes utilize different permeation pathways at rat P2X7 receptors

Activation of membrane P2X(7) receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-d-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-d-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X(7) receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X(7) receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-d-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X(7) receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-d-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-d-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway.

A basic peptide within the juxtamembrane region is required for EGF receptor dimerization

The epidermal growth factor receptor (EGFR) is fundamental for normal cell growth and organ development, but has also been implicated in various pathologies, notably tumors of epithelial origin. We have previously shown that the initial 13 amino acids (P13) within the intracellular juxtamembrane region (R 645–R 657) are involved in the interaction with calmodulin, thus indicating an important role for this region in EGFR function. Here we show that P13 is required for proper dimerization of the receptor. We expressed either the intracellular domain of EGFR (TKJM) or the intracellular domain lacking P13 (ΔTKJM) in COS-7 cells that express endogenous EGFR. Only TKJM was immunoprecipitated with an antibody directed against the extracellular part of EGFR, and only TKJM was tyrosine phosphorylated by endogenous EGFR. Using SK-N-MC cells, which do not express endogenous EGFR, that were stably transfected with either wild-type EGFR or recombinant full-length EGFR lacking P13 demonstrated that P13 is required for appropriate receptor dimerization. Furthermore, mutant EGFR lacking P13 failed to be autophosphorylated. P13 is rich in basic amino acids and in silico modeling of the EGFR in conjunction with our results suggests a novel role for the juxtamembrane domain (JM) of EGFR in mediating intracellular dimerization and thus receptor kinase activation and function.