Intracellular Juxtamembrane Research Articles

Role of EGFR Juxtamembrane Domain in EGFR Activation

The intracellular juxtamembrane domain of several receptor tyrosine kinases (RTKs) has been proposed to play an autoinhibitory role. The epidermal growth factor receptor (EGFR) has been widely used as a model in studies of the RTK activation mechanism. Extensive efforts have been devoted to studying the functions of EGFR's different domains; however, the role of the juxtamembrane domain in EGFR phosphorylation and activation is yet to be clarified. In this work, we replaced the 30 amino acids (674-704) of the EGF juxtamembrane domain with ten repeats of Glycine-Glycine-Serine (GGS). Preliminary results from western blots showed decreased phosphorylation of Tyr1148 in the EGFR-GGS chimeric receptor, which suggests that the juxtamembrane domain of EGFR plays an activating role rather than an inhibitory one. Currently we are using ligand titration and Foster resonance energy transfer (FRET) experiments to further explore the difference between wild type EGFR and the EGFR-GGS chimeric receptor. Results from this work will yield further insights into the function of the juxtamembrane domain in the EGFR activation process.

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.

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474-7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.

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.

Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment

Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation.

EGFR Juxtamembrane Domain, Membranes, and Calmodulin: Kinetics of Their Interaction

Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epidermal growth factor receptor (EGFR). The basic JMD also binds to acidic lipids in the inner leaflet of the plasma membrane, and this interaction may contribute an extra level of autoinhibition to the receptor. Binding of a ligand to the EGFR produces a rapid increase in intracellular calcium, [Ca 2+] i, and thus Ca/CaM. How does Ca/CaM compete with the plasma membrane for the JMD? Does Ca/CaM directly pull the JMD off the membrane or does Ca/CaM only bind to the JMD after it has dissociated spontaneously from the bilayer? To answer this question, we studied the effect of Ca/CaM on the rate of dissociation of fluorescent JMD peptides from phospholipid vesicles by making kinetic stop-flow measurements. Ca/CaM increases the rate of dissociation: an analysis of the differential equations that describe the dissociation shows that Ca/CaM must directly pull the basic JMD peptide off the membrane surface. These measurements lead to a detailed atomic-level mechanism for EGFR activation that reconciles the existence of preformed EGFR dimers/oligomers with the Kuriyan allosteric model for activation of the EGFR kinase domains.

The Intracellular Juxtamembrane Domain of the Epidermal Growth Factor (EGF) Receptor Is Responsible for the Allosteric Regulation of EGF Binding

We have previously shown that the binding of epidermal growth factor (EGF) to its receptor can best be described by a model that involves negative cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112-117). However, despite the fact that biochemical analyses indicate that EGF induces dimerization of its receptor, the binding data provided no evidence for positive linkage between EGF binding and dimer assembly. By analyzing the binding of EGF to a number of receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is positively linked to receptor dimerization but the linkage is abolished upon autophosphorylation of the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit negative cooperativity, indicating that mechanistically, cooperativity is distinct from the phenomenon of linkage. Nonetheless, both the positive linkage and the negative cooperativity observed in EGF binding require the presence of the intracellular juxtamembrane domain. This indicates the existence of inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 19238-19243). Our experiments expand the role of this domain to include the allosteric control of ligand binding by the extracellular domain.

Palmitoylation of the EGF Receptor Impairs Signal Transduction and Abolishes High-Affinity Ligand Binding

The intracellular juxtamembrane domain of the EGF receptor has been shown to be involved in the stimulation of the receptor's tyrosine kinase activity. To further explore the function of this portion of the EGF receptor, a consensus site for protein palmitoylation was inserted at the beginning of the juxtamembrane domain of the receptor. The altered EGF receptor incorporated [(3)H]palmitate, demonstrating that it was palmitoylated. Compared to the wild-type EGF receptor, the palmitoylated EGF receptor was significantly impaired in EGF-stimulated receptor autophosphorylation as well as ligand-induced receptor internalization. While both the wild-type and the palmitoylated EGF receptors exhibited a similar propensity to associate with lipid rafts, only the wild-type receptor exited lipid rafts in response to EGF. Binding of [(125)I]EGF to the wild-type EGF receptor showed a curvilinear Scatchard plot with both high- and low-affinity forms of the receptor. By contrast, the palmitoylated receptor exhibited only low-affinity EGF binding. These data suggest that the cytoplasmic juxtamembrane domain is involved not only in the transmission of the proliferative signal generated by ligand binding but also in facilitating the adoption of the high-affinity conformation by the extracellular ligand binding domain.

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.

Phosphorylation of Thr 654 but not Thr 669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding

Calcium-calmodulin (CaM) binding to the epidermal growth factor receptor (EGFR) has been shown to both inhibit and stimulate receptor activity. CaM binds to the intracellular juxtamembrane (JM) domain (Met 645–Phe 688) of EGFR. Protein kinase C (PKC) mediated phosphorylation of Thr 654 occurs within this domain. CaM binding to the JM domain inhibits PKC phosphorylation and conversely PKC mediated phosphorylation of Thr 654 or Glu substitution of Thr 654 inhibits CaM binding. A second threonine residue (Thr 669) within the JM domain is phosphorylated by the mitogen-activated protein kinase (MAPK). Previous results have shown that CaM interferes with EGFR-induced MAPK activation. If and how phosphorylation of Thr 669 affects CaM-EGFR interaction is however not known.In the present study we have used surface plasmon resonance (BIAcore) to study the influence of Thr 669 phosphorylation on real time interactions between the intracellular juxtamembrane (JM) domain of EGFR and CaM. The EGFR-JM was expressed as GST fusion proteins in Escherichia coli and phosphorylation was mimicked by generating Glu substitutions of either Thr 654 or Thr 669. Purified proteins were coupled to immobilized anti-GST antibodies at the sensor surface and increasing concentration of CaM was applied. When mutating Thr 654 to Glu 654 no specific CaM binding could be detected. However, neither single substitutions of Thr 669 (Gly 669 or Glu 669) nor double mutants Gly 654/Gly 669 or Gly 654/Glu 669 influenced the binding of CaM to the EGFR-JM. This clearly shows that PKC may regulate EGF-mediated CaM signalling through phosphorylation of Thr 654 whereas phosphorylation of Thr 669 seems to play a CaM independent regulatory role. The role of both residues in the EGFR-calmodulin interaction was also studied in silico. Our modelling work supports a scenario where Thr 654 from the JM domain interacts with Glu 120 in the calmodulin molecule. Phosphorylation of Thr 654 or Glu 654 substitution creates a repulsive electrostatic force that would diminish CaM binding to the JM domain. These results are in line with the Biacore experiments showing a weak binding of the CaM to the JM domain with Thr 654 mutated to Glu. Furthermore, these results provide a hypothesis to how CaM binding to EGFR might both positively and negatively interfere with EGFR-activity.

The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor‐like kinase

The rice gene Xa21 confers resistance against Xanthomonas oryzae pv. oryzae (Xoo). Xa21 encodes a receptor-like kinase (XA21). We demonstrate that XA21 autophosphorylates residues Ser686, Thr688 and Ser689 in vitro. Substitution of these residues with alanines did not affect the autophosphorylation function of this kinase, but specifically destabilized the resistance protein in vitro and in vivo. Plants carrying these same substitutions in XA21 were compromised in their resistance to the normally avirulent Xoo Philippine race 6. Additionally, we show that wild-type XA21 and the kinase-dead mutant with the invariable Lys736 residue mutated to glutamic acid were also proteolytically degraded in protein extracts. Finally, we show a correlation between the in vitro degradation and in vivo instability of the proteins. We propose that autophosphorylation of Ser686, Thr688 and Ser689 functions to stabilize XA21 against the developmentally controlled proteolytic activity.

Electrostatic interactions of peptides flanking the tyrosine kinase domain in the epidermal growth factor receptor provides a model for intracellular dimerization and autophosphorylation

The mechanism by which ligand-activated EGFR induces autophosphorylation via dimerization is not fully understood. Structural studies have revealed an extracellular loop mediated receptor dimerization. We have previously presented experimental data showing the involvement of a positive 13 amino acid peptide (R645-R657; P13+) from the intracellular juxtamembrane domain (JM) of EGFR important for intracellular dimerization and autophosphorylation. A model was presented that suggest that P13+ interacts with a negative peptide (D979-E991; P13-) positioned distal to the tyrosine kinase domain in the opposite EGFR monomer. The present work shows additional data strengthening this model. In fact, by analyzing protein sequences of 21 annotated ErbB proteins from 9 vertebrate genomes, we reveal the high conservation of peptides P13+ and P13- with regard to their sequence as well as their position relative to the tyrosine kinase (TK) domain. Moreover in silico structure modeling of these ErbB intracellular domains supports a general electrostatic P13+/P13- interaction, implying that the C-terminal of one receptor monomer is facing the TK domain of the other monomer in the receptor dimer and vice versa. This model provides new insights into the molecular mechanism of ErbB receptor activation and suggests a new strategy to pharmacologically interfering with ErbB receptor activity.

P-Cadherin Promotes Cell-Cell Adhesion and Counteracts Invasion in Human Melanoma

Malignant transformation of melanocytes frequently coincides with alterations in epithelial cadherin (E-cadherin) expression, switching on of neural cadherin (N-cadherin), and, when progressed to a metastatic stage, loss of membranous placental cadherin (P-cadherin). In vitro studies of melanoma cell lines have shown invasion suppressor and promoter roles for E-cadherin and N-cadherin, respectively. In the present study, we investigated the effect of P-cadherin on aggregation and invasion using melanoma cells retrovirally transduced with human P-cadherin. De novo expression of P-cadherin in P-cadherin-negative cell lines (BLM and HMB2) promoted cell-cell contacts and Ca2+-dependent cell-cell aggregation in two- and three-dimensional cultures, whereas it counteracted invasion. These effects were not observed following P-cadherin transduction of endogenously P-cadherin-positive MeWo cells. In addition, P-cadherin-transduced BLM cells coaggregated with keratinocytes and showed markedly reduced invasion in a reconstructed skin model. The proadhesive and anti-invasive effects of P-cadherin were abolished on targeted mutation of its intracellular juxtamembrane domain or its extracellular domain. For the latter mutation, we mimicked a known missense mutation in P-cadherin (R503H), which is associated with congenital hypotrichosis with juvenile macular dystrophy.

Novel Germline Mutation of KIT Associated With Familial Gastrointestinal Stromal Tumors and Mastocytosis

Gastrointestinal stromal tumors (GISTs) are often associated with activating KIT mutations, affecting regulatory domains of the KIT tyrosine kinase. Sporadic mastocytosis in adults is usually also caused by KIT mutations that, however, activate KIT by affecting the intracellular enzymatic site of the molecule. Most GISTs respond to KIT inhibitors that bind to the enzymatic site; in most cases of mastocytosis, however, the modified enzymatic site is not affected by these drugs. We present a kindred with both familial GISTs and mastocytosis that express a novel germline KIT mutation in exon 8, resulting in deletion of codon 419 and affecting the extracellular domain of KIT. This mutation activates KIT, and the mutant KIT is inhibited by the tyrosine kinase inhibitor imatinib mesylate. Our studies identify a new regulatory region in the KIT molecule and strongly suggest that patients with extracellular KIT mutations respond to tyrosine kinase inhibitors.

Analysis of Human Multidrug Resistance Protein 1 (ABCC1) by Matrix-Assisted Laser Desorption Ionization/Time of Flight Mass Spectrometry: Toward Identification of Leukotriene C4Binding Sites

Multidrug resistance in tumor cells may be caused by reduced drug accumulation resulting from expression of one or more proteins belonging to the ATP-binding cassette (ABC) transporter superfamily. In addition to their drug efflux properties, certain ABC proteins such as multidrug resistance protein 1 (MRP1) (ABCC1) mediate the ATP-dependent transport of a broad array of organic anions. The intrinsically photoreactive glutathione-conjugated cysteinyl leukotriene C4 (LTC4) is a high-affinity physiological substrate of MRP1 and is widely regarded as a model compound for evaluating the substrate binding and transport properties of wild-type and mutant forms of the transporter. In the present study, we have optimized high-level expression of recombinant human MRP1 in Pichia pastoris and developed a two-step purification scheme that results in purification of the transporter to >90% homogeneity. Peptide mapping by matrix-assisted laser desorption ionization/time of flight mass spectrometry of the peptides generated by in-gel protease digestions of purified underglycosylated MRP1 identified 96.7% of the MRP1 sequence with >98% coverage of its 17 transmembrane helices. Subsequent comparisons with mass spectra of MRP1 photolabeled with LTC4 identified six candidate LTC4-modified peptide fragments that are consistent with the conclusion that the intracellular juxtamembrane positions of transmembrane helices 6, 7, 10, 17, and a COOH-proximal portion of the cytoplasmic loop that links the first and second membrane spanning domains are part of the LTC4 binding site of the transporter. Our studies confirm the usefulness of mass spectrometry for analysis of mammalian polytopic membrane proteins and for identification of substrate binding sites of human MRP1.

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 Study of Mutations in the c-kit Gene of 32 Dogs with Mastocytoma

Mutations in the intracellular juxtamembrane domain of the c-kit gene in 32 dogs with different grades of histologically confirmed mastocytoma were studied. Transcript RNAs extracted from neoplastic tissue surgically collected from dogs of different breeds and from a negative control were reverse transcribed into complementary DNA and amplified by polymerase chain reaction. The region corresponding to the c-kit juxtamembrane domain was sequenced and compared with GenBank sequences. Two different types of mutations were identified within exon 11: a previously underscribed single-nucleotide substitution and a 6-bp deletion. The c-kit juxtamembrane domain sequences of all dogs were grouped in 3 clusters. No mutations were detected in tissues constitutively expressing c-kit (cerebellum and spleen), obtained from dogs not affected by mastocytoma (controls). All the substitutions were found in dogs bearing grade I or II mast cell tumors; the deletion was detected in 1 dog with grade II mastocytoma.