Membrane-proximal Cytoplasmic Domain Research Articles

αIIbβ3 variants in ten families with autosomal dominant macrothrombocytopenia: Expanding the mutational and clinical spectrum.

BackgroundRare pathogenic variants in either the ITGA2B or ITGB3 genes have been linked to autosomal dominant macrothrombocytopenia associated with abnormal platelet production and function, deserving the designation of Glanzmann Thrombasthenia-Like Syndrome (GTLS) or ITGA2B/ITGB3-related thrombocytopenia.ObjectivesTo describe a series of patients with familial macrothrombocytopenia and decreased expression of αIIbβ3 integrin due to defects in the ITGA2B or ITGB3 genes.MethodsWe reviewed the clinical and laboratory records of 10 Portuguese families with GTLS (33 patients and 11 unaffected relatives), including the functional and genetic defects.ResultsPatients had absent to moderate bleeding, macrothrombocytopenia, low αIIbβ3 expression, impaired platelet aggregation/ATP release to physiological agonists and low expression of activation-induced binding sites on αIIbβ3 (PAC-1) and receptor-induced binding sites on its ligand (bound fibrinogen), upon stimulation with TRAP-6 and ADP. Evidence for constitutive αIIbβ3 activation, occurred in 2 out of 9 patients from 8 families studied, but also in 2 out of 12 healthy controls. We identified 7 missense variants: 3 in ITGA2B (5 families), and 4 in ITGB3 (5 families). Three variants (αIIb: p.Arg1026Trp and p.Arg1026Gln and β3: p.Asp749His) were previously reported. The remaining (αIIb: p.Gly1007Val and β3: p.Thr746Pro, p.His748Pro and p.Arg760Cys) are new, expanding the αIIbβ3 defects associated with GTLS. The integration of the clinical and laboratory data allowed the identification of two GTLS subgroups, with distinct disease severity.ConclusionsPreviously reported ITGA2B and ITGB3 variants related to thrombocytopenia were clustered in a confined region of the membrane-proximal cytoplasmic domains, the inner membrane clasp. For the first time, variants are reported at the outer membrane clasp, at the transmembrane domain of αIIb, and at the membrane distal cytoplasmic domains of β3. This is the largest single-center series of inherited macrothrombocytopenia associated with αIIbβ3 variants published to date.

Utilizing CRISPR-CAS9 Gene Editing Technology in Human Pluripotent Stem Cells to Study Platelet Integrin αIIbβ3 Function

Integrins are a family of cell surface receptors that transmit signals bidirectionally across plasma membranes. Cellular agonists induce 'inside-out' signals that cause ligand binding to integrins; in turn, this causes integrin-mediated 'outside-in' signals that control cellular functions such as adhesion and migration. The platelet integrin αIIbβ3 has served as a paradigm for deciphering these bidirectional signaling mechanisms. Moreover, site-directed mutagenesis has been essential for dissecting integrin structure-function relationships. For platelet integrins such as αIIbβ3, the structure-function consequences of the site-directed mutations have previously been studied in epithelial cell lines, such as Chinese hamster ovary (CHO) cells. Only recently has technology advanced sufficiently to enable study of platelet integrins in their normal cellular environment, the platelet-producing megakaryocyte. The advent of human induced pluripotent stem cells (iPSC), coupled with the ability to gene edit these cells using the CRISPR-CAS9 technology provides a way to study αIIbβ3 mutants in megakaryocytes. Following platelet stimulation, αIIbβ3 rapidly undergoes a global rearrangement in which a clasp composed of its extracellular stalk, transmembrane, and membrane-proximal cytoplasmic domains is disrupted causing the αIIbβ3 headpiece to open exposing a ligand binding site. Using computational methods, we predicted mutations that would destabilize the stalk domain, thereby causing αIIbβ3 activation. These predictions were confirmed by showing that the mutations were indeed able to cause constitutive αIIbβ3 activation in CHO cells (Donald et al, J. Biol. Chem. 2010; Tan et al, Biochemistry 2019). To translate these findings to human cells and compare the functional consequences of the mutations when endogenously expressed in iPSC-derived megakaryocytes, we chose a highly activating V760A missense mutation located in the stalk region of the αIIb subunit. Using an established control iPSC line designated CHOPWT14, both heterozygous and homozygous V760A missense mutations were created in the ITGA2B gene encoding αIIb using our CRISPR-CAS9 protocol (Maguire et al. Current Protocols in Stem Cell 2019). The generation of these mutant cell lines from a genetically identical control cell line eliminates the heterogeneity and variability in downstream applications that was observed when using lines of different genetic backgrounds. To ensure genetic identity of the parent and mutant lines, copy number variation or CNV analyses were performed and shown to be identical in all lines. To study ligand binding of αIIbβ3 expressing the V760A mutation in iPSC-derived megakaryocytes, the isogenic control and mutant lines were differentiated to blood using our serum- and stromal-free directed differentiation protocol (Mills et al. Methods in Mol. Biol. 2014). Briefly, the differentiation of human iPSCs to blood follows primitive hematopoiesis which recapitulates the developmental stages observed in the embryo of primitive streak formation, mesoderm specification, and hematopoietic development, including megakaryocytes. To measure conformational change in αIIbβ3, the activation-dependent monoclonal antibody PAC-1 was used as a surrogate for fibrinogen. By analyzing CD41+CD42b+ megakaryocytes by flow cytometry, PAC-1 was shown to bind constitutively to 23.4% and 26.0% of the megakaryocytes expressing heterozygous and homozygous V760A mutations, respectively, compared to 9.04% of control megakaryocytes. The specificity of PAC-1 binding was confirmed by a reduction in binding to <1% following pretreatment with EDTA. In addition, thrombin stimulation increased PAC-1 binding to >65% in all lines, indicating normal overall αIIbβ3 function. Essentially identical results were obtained when FITC-fibrinogen was used instead of PAC-1. These data show that 1) structure-function studies of computationally identified mutations confirmed in CHO cells can be analyzed using human iPSC-derived megakaryocytes, 2) mutations shown to be highly active in CHO cells appear to be constrained or less constitutively active in human megakaryocytes, and 3) more in-depth analyses of platelet integrin structure-function relationships will be possible using human megakaryocytes. Disclosures No relevant conflicts of interest to declare.

Dematin and Adducin Tether Sodium-Hydrogen Exchanger, NHE1, to Erythrocyte Membrane Cytoskeleton

Dematin is a critical component of the membrane junctional complex in red blood cells. It tethers the spectrin cytoskeleton proteins to the membrane and its genetic deletion in mice causes dissociation of the spectrin, actin and β-adducin from the membrane resulting in the collapse of the red blood cells (RBCs). As dematin lacks a transmembrane domain, it is still unclear how this critical component of the junctional complex is anchored to the RBC membrane. Our previous studies have shown that the multi-transmembrane glucose transporter-1 (GLUT1) interacts with dematin and β-adducin in human RBCs, suggesting a potential role for GLUT1 in recruiting dematin to the membrane. However, as mouse RBCs do not express a GLUT1 homologue, an equivalent membrane receptor for dematin and/or adducin in mice remains to be determined. Using multiple in vitro and in vivo biochemical assays, here we demonstrate that the ubiquitously expressed plasma membrane Na+/H+ exchanger, NHE1 (Slc9a1), is one of the receptors for dematin and β-adducin in mature mouse red blood cells. NHE1 directly interacts with the core domain of dematin. Moreover, the dematin headpiece domain mutant S381E, which binds to the core domain with a higher affinity than the wild type, abolished the biochemical interaction between dematin and NHE1. This observation suggests that NHE1 and dematin headpiece domain compete for the same binding site(s) on the core domain. Furthermore, this finding highlights a molecular mechanism whereby an intermolecular switch of dematin regulates its interaction with NHE1 by phosphorylation. Dematin and β-adducin directly interact with NHE1 at its membrane-proximal cytoplasmic domain, which in turn regulates NHE1 activity in response to growth factor stimuli and intracellular pH alterations. Accordingly,we observed an increased cellular sodium content in erythrocytes of dematin headpiece and adducin double knockout mice (DAKO), suggesting a higher NHE1 activity in DAKO erythrocytes. Unlike GLUT1, NHE1 is expressed in both mouse and human RBCs. Thus, our results provide a novel mechanism for linking NHE1 to membrane skeleton and multiple cell signaling pathways through dematin and adducin (Figure 1). Since NHE1 is one of the major regulators of intracellular pH and hypertonic stress, our findings raise the possibility that the dematin-adducin-NHE1 complex may modulate these functions in RBCs as well as in other cell types with broad impact on the regulation of the actin cytoskeleton and cell migration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of an arginine–lysine rich motif in maturation and trafficking of CD19

Normal expression of CD19 on the surface of B cells requires the presence of the tetraspanin molecule CD81. Previous studies have shown that surface expression of CD19 is highly reduced in CD81-deficient mouse B cells and that it is completely absent in an antibody deficient human patient with a mutation in the CD81 gene. The current study explored the contribution of an arginine–lysine rich motif, present in the membrane-proximal cytoplasmic domain of CD19, for the maturation and trafficking of this molecule. We demonstrate that this motif plays a role in the maturation and recycling of CD19 but in a CD81-independent manner.

Palmitoylated peptide, being derived from the carboxyl-terminal sequence of the integrin αIIb cytoplasmic domain, inhibits talin binding to αIIbβ3

The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal 1000LEEDDEEGE1008 region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000–1008) inhibits platelet aggregation induced by thrombin or by pal-K-989–995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain 989KVGFFKR995. We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-1000LEEDDE1005 (pal-K-1000–1005) and palmitoyl-K-1005EEGE1008 (pal-K-1005–1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000–1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000–1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000–1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3.

Identification of a Region within the Cytoplasmic Domain of Sema4D That Binds Calmodulin and Regulates Shedding of the Sema4D Exodomain in Platelets,

Abstract 3249We have previously shown that the 150 kDa semaphorin family member, Sema4D (or CD100), is expressed on the surface of human and mouse platelets, where it is able to selectively reinforce collagen-initiated platelet activation by engaging receptors in trans on adjoining platelets in a contact-dependent manner. Key to this effect is the Sema4D extracellular domain, which in addition to being a ligand for Sema4D receptors, is a substrate for the metalloprotease, ADAM17 in platelets. Our previous studies suggest that ADAM17 cleaves Sema4D and other platelet surface proteins close to the platelet plasma membrane gradually producing, in the case of Sema4D, a single large (≈120 kDa) exodomain fragment and a smaller (≈28 kDa) fragment that includes the transmembrane domain and the cytoplasmic domain and remains associated with the platelet (Zhu, et al., PNAS 2007). Exodomain shedding in platelets can be triggered by the phorbol ester, PMA, and by physiologic agonists such as thrombin that raise the cytoplasmic Ca++ concentration, but the mechanisms that regulate the shedding of Sema4D have not been defined. Here we have studied the potential role of an interaction between calmodulin and the Sema4D cytoplasmic domain. Using a public resource (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html), we identified a potential calmodulin-binding sequence (GYLPRQCLKFRSALLIGKKKPKS-COOH, Gly758–Ser780) within the membrane-proximal region of the Sema4D cytoplasmic domain. To test whether this region binds calmodulin, a 23 amino acid peptide corresponding to the predicted Sema4D calmodulin binding sequence (SCBP) was synthesized, as was a scrambled control peptide (RLIKACRQPKPKYKLLGFGSSKL or scrambled SCBP), which is not predicted to bind calmodulin. The results show that SCBP, but not scrambled SCBP, was able to bind to calmodulin-agarose and retrieve calmodulin from platelet lysates. As constitutive association of calmodulin with glycoprotein (GP) Ib has been shown prevent ADAM17-dependent GPIb alpha shedding in platelets, we incubated human platelets with the calmodulin inhibitor, W7. The inhibitor induced gradual Sema4D shedding that was detectable after 5 min and reached a maximum at 60 min, kinetics that are similar to those we have observed with platelet agonists. However, in contrast to platelet agonists, W7-induced Sema4D shedding generated a smaller retained fragment (≈24 kDa Vs. 28 kDa) suggesting that there is either a second or different site of cleavage. Despite their polybasic sequences, flow cytometry and confocal microscopy showed that FITC-conjugated SCBP and scrambled-SCBP are able to cross the plasma membrane. Addition of SCBP, but not scrambled-SCBP, to platelet caused cleavage of Sema4D, producing the same 28 kDa fragment observed with thrombin and PMA. In all cases cleavage of Sema4D was blocked by the metalloprotease inhibitor, TAPI-2. Combined with our earlier observations, these results suggest that 1) Sema4D is a calmodulin binding protein with a site of interaction in the membrane-proximal cytoplasmic domain and a site of cleavage by ADAM17 in the membrane-proximal exodomain, 2) the detachment of calmodulin from Sema4D may be the trigger for Sema4D cleavage in response to platelet agonists, and 3) in contrast to W7, decoying calmodulin from binding sites on Sema4D and other metalloprotease substrates on the platelet surface, as we have done here with a Sema4D cytoplasmic domain peptide, may trigger the same events seen in activated platelets and provide a tool to understand the underlying mechanisms. Disclosures:No relevant conflicts of interest to declare.

Identification of Interacting Hot Spots in the β3 Integrin Stalk Using Comprehensive Interface Design

Protein-protein interfaces are usually large and complementary surfaces, but specific side chains, representing energetic "hot spots," often contribute disproportionately to binding free energy. We used a computational method, comprehensive interface design, to identify hot spots in the interface between the stalk regions of the β3 and the complementary αIIb and αv integrin subunits. Using the Rosetta alanine-scanning and design algorithms to predict destabilizing, stabilizing, and neutral mutations in the β3 region extending from residues Lys(532) through Gly(690), we predicted eight alanine mutations that would destabilize the αIIbβ3 interface as well as nine predicted to destabilize the αvβ3 interface, by at least 0.3 kcal/mol. The mutations were widely and unevenly distributed, with four between residues 552 and 563 and five between 590 and 610, but none between 565 and 589, and 611 and 655. Further, mutations destabilizing the αvβ3 and αIIbβ3 interfaces were not identical. The predictions were then tested by introducing selected mutations into the full-length integrins expressed in Chinese hamster ovary cells. Five mutations predicted to destabilize αIIb and β3 caused fibrinogen binding to αIIbβ3, whereas three of four predicted to be neutral or stabilizing did not. Conversely, a mutation predicted to destabilize αvβ3, but not αIIbβ3 (D552A), caused osteopontin binding to αvβ3, but not fibrinogen binding to αIIbβ3. These results indicate that stability of the distal stalk interface is involved in constraining integrins in stable, inactive conformations. Further, they demonstrate the ability of comprehensive interface design to identify functionally significant integrin mutations.

Disruption of an env tyrosine-dependent sorting signal does not affect susceptibility of HIV-1 to cytotoxic T lymphocytes

Infections with live-attenuated viruses within SIV models of HIV-1 pathogenesis have offered the best evidence that a vaccine could generate protective immunity. Desrosiers and colleagues [1] first demonstrated this principle using nef-defective SIV, which established a chronic low-grade asymptomatic infection in macaques associated with protection against subsequent challenge with wild-type SIV. At least in part, this phenomenon appeared to be due to attenuation of viral replication. More recently, Shacklett et al [2] and Hoxie and et al [3] have examined attenuated SIV infection via mutations in the transmembrane domain of gp41, which also subsequently protects from challenge by wild-type SIV. Shacklett et al found that SIV containing multiple (stop and point) mutations disrupting this domain reduced viral replicative capacity in vitro, likely due to effects on gp41 membrane trafficking in infected cells [4, 5]. Hoxie and colleagues more specifically created mutations in the tyrosine-based sorting motif (Y712xxϕ) in the membrane-proximal cytoplasmic domain of gp41 [3]. Despite the role this motif in gp41 trafficking [4, 5], the mutations appeared to have minimal impact on viral fitness, reflected by normal peak viremia during acute infection in vivo and growth kinetics in vitro. Thus the mechanism of attenuation and subsequent immune protection is unclear, but probably not due to markedly reduced viral replication capacity. Interestingly, in vivo CD8 depletion experiments have suggested that CD8+ T lymphocytes (CTLs) may contribute to the protective immunity [6]. To assess whether analogous mutations in HIV-1 might affect viral susceptibility to CTLs, mutations in the Y712xxϕ motif were constructed in HIV-1 NL4-3 [7] by point mutagenesis (QuikChange, Stratagene). These included EnvY712I, EnvY712S and EnvΔGY mutations in the cytoplasmic domain of gp41 (Figure 1A), engineered into the whole genome context of NL4-3.1 containing the clade B consensus sequence at Gag 77-85 (HXB2 a.a. numbering) [8]. These viruses were examined for their ability to replicate in T1 cells [9] (Figure 1B). The EnvY712I and EnvY712S mutants had growth kinetics similar to wild-type NL4-3 (EnvY712), similar to SIVmac239 [3]. In contrast, the EnvΔGY mutant was replication incompetent, suggesting that this mutation more severely impaired HIV-1 than SIV. Figure 1 Susceptibility of Env Y712xxϕ motif mutant viruses to HIV-1-specific CTL To assess the susceptibility of these mutants to CTLs, these viruses were tested in virus suppression assays [10, 11] using HIV-1-specific CTL clones: S1-SL9-3.23T recognizing the HLA A*02-restricted epitope SLYNTVATL in Gag p17 (a.a. 77-85) [12], S31-IV9-10.11T recognizing the HLA A*02-restricted epitope ILKEPVHGV in RT (a.a. 309-317), and S58-PL10-10.8 recognizing the HLA A*02-restricted epitope PLTFGWCYKL in Nef (a.a. 136-145). The HIV-1-permissive target cells were A*02-expressing T1 cells [10]. Comparisons of these viruses showed similar degrees of suppression of the mutant and wild-type viruses by the three CTL clones (Figure 1C and D). Similar results were noted in independent experiments using two other CTL clones recognizing other epitopes (not shown). Overall, these results suggested that directly increased susceptibility to CTL inhibition is not the mechanism of in vivo attenuation of infection with viruses containing these mutations. Because attenuated SIV infection has been the most robust example of protective vaccination in the SIV-macaque model [1, 6, 13–15], understanding how viral attenuation affects antiviral immunity is clearly an important goal. The observation that SIV mutated in the Y712xxϕ motif yields infections with typical high peak viremia followed by chronic low viremia in macaques subsequently protected from wild-type virus challenge [2, 3, 6] suggests that viral replication is not markedly affected by the mutations, but that replication is suppressed during chronic infection after development of CTL responses, which are the major determinant of set-point viremia [16–18]. Furthermore, preliminary data suggests that the low set-point viremia of macaques infected with Y712xxϕ-disrupted SIV may be related to the CTL response [6]. Thus, a simple explanation could be that disruption of this motif somehow renders SIV directly more susceptible to CTL. However, our results suggest that this is not the case for HIV-1; disruption of the Y712xxϕ motif did not appear to increase susceptibility of HIV-1 to CTLs directly. While our in vitro assay may not necessarily predict the interaction of virus and CTLs in vivo, it seems likely that the mechanism of attenuation is either not mediated by CTLs, or indirectly increases the antiviral activity of CTLs. Interestingly, it has been suggested that the HIV-1 Env may facilitate viral escape from CTLs in lymph nodes [19, 20]. Although our data do not address this issue directly, they are compatible with a mechanism whereby reduced levels of Env could reduce viral escape from CTLs. Further work would be required to explore this possibility.

A palmitoylated peptide, derived from the acidic carboxyl-terminal segment of the integrin αIIb cytoplasmic domain, inhibits platelet activation

Platelet integrin αIIbβ3 contains an acidic membrane distal motif, 1000LEEDDEEGE1008, in the cytoplasmic domain of the αIIb subunit. We showed that a lipid-modified peptide corresponding to the above region, palmitoyl-K-LEEDDEEGE (pal-K-1000-1008), is platelet permeable and has inhibited platelet aggregation induced by 0.4 U/ml of thrombin (IC50 = 164 µM). Moreover the peptide inhibited both Fibrinogen and PAC-1, binding to activated platelets. The non palmitoylated analog was inactive. A modified, scrambled acidic peptide (palmitoyl-K-GDDEELEEE), showed significant lower inhibitory activity than pal-K-1000-1008. A palmitoylated peptide corresponding to the membrane proximal cytoplasmic domain of αIIb, 989KGVFFKR995 (pal-989-995), is known to specifically induce platelet aggregation. Pal-K-1000-1008 was an inhibitor of human washed platelet aggregation induced by pal-K-989-995 (IC50 = 15 µM). Moreover, pal-K-1000-1008 inhibited phosphorylation of ERK and FAK, two protein kinases involved in platelet activation and aggregation. Our results favour the assumption that the interaction of the membrane proximal sequence 989KGVFFKR995 of the cytoplasmic domain of αIIb with the acidic terminal 1000LEEDDEEGE1008 motif may be an important structural factor in platelet signaling, leading to platelet activation and aggregation.

Membrane-proximal cytoplasmic domain of Moloney murine leukemia virus envelope tail facilitates fusion

Removal of the R peptide (residues 617–632) from the Moloney murine leukemia virus (MoMuLV) envelope protein (Env) cytoplasmic tail potentiates fusion. We examined the role of the membrane-proximal cytoplasmic domain (598–616) of the MoMuLV Env in the Env-mediated membrane fusion and incorporation. The Env truncated at 616 exhibits maximum fusogenicity in cell-to-cell fusion assay. By comparison, full tail Env (632) and the Env truncated to residue 601 mediated fusion at 40%. The Envs truncated to residues 598 or 595 are not fusogenic. Progressive cytoplasmic tail truncation correlated with decreased Env incorporation into virions. Substitution of the domain 598–616 with an amphiphilic α-helix from melittin results in maximally fusogenic Envs that efficiently incorporated into transduction competent virions. However, substitution of the domain 598–616 with random or hydrophilic sequences caused loss of the Env fusogenicity and titer while retaining incorporation. Further, a secondary structure prediction analysis of 27 unrelated Env cytoplasmic tails indicates a common (23/27) propensity for an amphiphilic α-helical domain at immediate proximity to the viral membrane. These results support the suggestion that viral fusion is enhanced by a membrane-proximal cytoplasmic amphiphilic α-helix in Env tail. The model of its action is proposed.

Membrane activity of an amphiphilic α-Helical membrane-proximal cytoplasmic domain of the MoMuLV envelope glycoprotein

In the Moloney murine leukemia virus (MoMuLV) envelope glycoprotein (Env) we identified a membrane-proximal cytoplasmic domain (residues 598–616) that facilitates the Env incorporation into virions and Env-mediated fusion [Rozenberg, Y., Conner, J., Aguilar-Carreno, H., Chakraborti, S., Dimiter, D.S., Anderson, W.F., 2008. Viral entry: membrane-proximal cytoplasmic domain of MoMuLV envelope tail facilitates fusion. In the same issue. (accompanying paper)]. By biophysical methods (CD, EPR) a corresponding peptide (membrane-proximal peptide, 598–616) was demonstrated to form a membrane-parallel amphiphilic α-helix in the presence of membranes. Electrophysiological studies with planar bilayers and liposomes indicate that the membrane-proximal peptide is membrane destabilizing. This peptide and the fusion peptide from the MoMuLV transmembrane (TM) ectodomain were tested for their effect on the bilayer for hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine (T H). Importantly, the external fusion peptide and the internal membrane-proximal peptides of MoMuLV env exert opposite effects on membrane curvature. The fusion peptide lowers T H while the membrane proximal peptide raises it. These effects on T H correlate with the ability of these peptides to induce lipid mixing in large unilamellar vesicles composed of dioleoylphosphatidylethanolamine: dioleoylphosphatidylcholine:cholesterol (1:1:1 mol). When added externally to preformed liposomes, the N-terminal fusion peptide promotes lipid mixing while the cytoplasmic membrane-proximal peptide inhibits this effect. These finding indicate a possible mechanism by which the membrane-proximal domain in MoMuLV Env may affect the formation of membrane fusion intermediates.

Activation of Platelet αIIbβ3 by an Exogenous Peptide Corresponding to the Transmembrane Domain of αIIb

A transmembrane domain heterodimer, acting in concert with a membrane-proximal cytoplasmic domain clasp, is thought to maintain integrins in a low affinity state. To test whether helix-helix interactions between the alphaIIb and beta3 transmembrane domains regulate the activity of integrin alphaIIbbeta3, we synthesized a soluble peptide corresponding to the alphaIIb transmembrane domain, designated alphaIIb-TM, and we studied its ability to affect alphaIIbbeta3 activity in human platelets. alphaIIb-TM was alpha-helical in detergent micelles and phospholipid vesicles, readily inserted into membrane bilayers, bound to intact purified alphaIIbbeta3, and specifically associated with the transmembrane domain of alphaIIb, rather than the transmembrane domains of beta3, alpha2, and beta1, other integrin subunits present in platelets. When added to suspensions of gel-filtered platelets, alphaIIb-TM rapidly induced platelet aggregation that was not inhibited by preincubating platelets with the prostaglandin E(1) or the ADP scavenger apyrase but was prevented by the divalent cation chelator EDTA. Furthermore, alphaIIb-TM induced fibrinogen binding to platelets but not the binding of osteopontin, a specific ligand for platelet alphavbeta3. The peptide also induced fibrinogen binding to recombinant alphaIIbbeta3 expressed by Chinese hamster ovary cells, confirming that its effect was independent of platelet signal transduction. Finally, transmission electron microscopy of purified alphaIIbbeta3 revealed that alphaIIb-TM shifted the integrin from a closed configuration with its stalks touching to an open configuration with separated stalks. These observations demonstrate that transmembrane domain interactions regulate integrin function in situ and that it is possible to target intra-membranous protein-protein interactions in a way that can have functional consequences.

Transmembrane Domain Helix Packing Stabilizes Integrin αIIbβ3 in the Low Affinity State

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.

Five Residues in the HtrI Transducer Membrane-proximal Domain Close the Cytoplasmic Proton-conducting Channel of Sensory Rhodopsin I

Transducer-free sensory rhodopsins carry out light-driven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous NpHtrII to interact with the E-F loop of its cognate receptor NpSRII in the accompanying article (Yang, C.-S., Sineshchekov, O., Spudich, E. N., and Spudich, J. L. (2004) J. Biol. Chem. 279, 42970-42976). These results strongly suggest that the membrane-proximal region of Htr proteins interact with their cognate sensory rhodopsin cytoplasmic domains as part of the signal-relay coupling between the proteins.

The Cytoplasmic Membrane-proximal Domain of the HtrII Transducer Interacts with the E-F Loop of Photoactivated Natronomonas pharaonis Sensory Rhodopsin II

The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Forster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Forster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.

Suramin Derivatives as Inhibitors and Activators of Protein-tyrosine Phosphatases

Protein-tyrosine phosphatases (PTPs) are important signaling enzymes that have emerged within the last decade as a new class of drug targets. It has previously been shown that suramin is a potent, reversible, and competitive inhibitor of PTP1B and Yersinia PTP (YopH). We therefore screened 45 suramin analogs against a panel of seven PTPs, including PTP1B, YopH, CD45, Cdc25A, VHR, PTPalpha, and LAR, to identify compounds with improved potency and specificity. Of the 45 compounds, we found 11 to have inhibitory potency comparable or significantly improved relative to suramin. We also found suramin to be a potent inhibitor (IC(50) = 1.5 microm) of Cdc25A, a phosphatase that mediates cell cycle progression and a potential target for cancer therapy. In addition we also found three other compounds, NF201, NF336, and NF339, to be potent (IC(50) < 5 microm) and specific (at least 20-30-fold specificity with respect to the other human PTPs tested) inhibitors of Cdc25A. Significantly, we found two potent and specific inhibitors, NF250 and NF290, for YopH, the phosphatase that is an essential virulence factor for bubonic plague. Two of the compounds tested, NF504 and NF506, had significantly improved potency as PTP inhibitors for all phosphatases tested except for LAR and PTPalpha. Surprisingly, we found that a significant number of these compounds activated the receptor-like phosphatases, PTPalpha and LAR. In further characterizing this activation phenomenon, we reveal a novel role for the membrane-distal cytoplasmic PTP domain (D2) of PTPalpha: the direct intramolecular regulation of the activity of the membrane-proximal cytoplasmic PTP domain (D1). Binding of certain of these compounds to PTPalpha disrupts D1-D2 basal state contacts and allows new contacts to occur between D1 and D2, which activates D1 by as much as 12-14-fold when these contacts are optimized.

CD4 Receptor Localized to Non-raft Membrane Microdomains Supports HIV-1 Entry: IDENTIFICATION OF A NOVEL RAFT LOCALIZATION MARKER IN CD4

Despite the preferential localization of CD4 to lipid rafts, the significance and role of these microdomains in HIV-1 entry is still controversial. The possibility that CD4, when localized to non-raft domains, might be able to support virus entry cannot be excluded. Because disintegration of rafts by extraction of cellular cholesterol with methyl-beta-cyclodextrin suffers from various adverse effects, we investigated molecular determinants controlling raft localization of the CD4 receptor. Extensive mutagenesis of the receptor showed that a raft-localizing marker, consisting of a short sequence of positively charged amino acid residues, RHRRR, was present in the membrane-proximal cytoplasmic domain of CD4. Substitution of the RHRRR sequence with alanine residues abolished raft localization of the CD4 mutant, RA5, as determined biochemically using solubilization in nonionic detergents and by confocal microscopy. The possible inhibitory effect of the introduced mutations on the adjacent CVRC palmitoylation site was ruled out because wild type (wt) CD4 and RA5, but not a palmitoylation-deficient mutant, were efficiently palmitoylated. Nonetheless, the RA5 mutant supported productive virus entry to levels equivalent to that of wild type (wt) CD4. Sucrose gradient analysis of Triton X-100 virus lysates showed that Gag and envelope gp120 proteins accumulated in low buoyant, high-density fractions. This pattern was changed after virus incubation with cells. Whereas Gag proteins localized to lipid rafts in cells expressing wt CD4 and RA5, gp120 accumulated in rafts in cells expressing wt CD4 but not RA5. We propose that raft localization of CD4 is not required for virus entry, however, post-binding fusion/entry steps may require lipid raft assembly.

The Cell Adhesion Molecule CEACAM1-L Is a Substrate of Caspase-3-mediated Cleavage in Apoptotic Mouse Intestinal Cells

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.

Multiple Signals Mediate Proliferation, Differentiation, and Survival from the Granulocyte Colony-stimulating Factor Receptor in Myeloid 32D Cells

Granulocyte colony-stimulating factor (G-CSF) regulates neutrophil production through activation of its cognate receptor, the G-CSF-R. Previous studies with deletion mutants have shown that the membrane-proximal cytoplasmic domain of the receptor is sufficient for mitogenic signaling, whereas the membrane-distal domain is required for differentiation signaling. However, the function of the four cytoplasmic tyrosines of the G-CSF-R in the control of proliferation, differentiation, and survival has remained unclear. Here we investigated the role of these tyrosines by expressing a tyrosine "null" mutant and single tyrosine "add back" mutants in maturation-competent myeloid 32D cells. Clones expressing the null mutant showed only minimal proliferation and differentiation, with survival also reduced at low G-CSF concentrations. Analysis of clones expressing the add-back mutants revealed that multiple tyrosines contribute to proliferation, differentiation, and survival signals from the G-CSF-R. Analysis of signaling pathways downstream of these tyrosines suggested a positive role for STAT3 activation in both differentiation and survival signaling, whereas SHP-2, Grb2 and Shc appear important for proliferation signaling. In addition, we show that a tyrosine-independent "differentiation domain" in the membrane-distal region of the G-CSF-R appears necessary but not sufficient for mediating neutrophilic differentiation in these cells.

Stimulation of Stat5 by Granulocyte Colony-Stimulating Factor (G-CSF) Is Modulated by Two Distinct Cytoplasmic Regions of the G-CSF Receptor

In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.