Human Β3 Subunit Research Articles

ATP1B3: a virus-induced host factor against EV71 replication by up-regulating the production of type-I interferons

Enterovirus 71 (EV71) infection can cause severe diseases, and is becoming increasingly common in children. In the current study, we carried out yeast two-hybrid assays to screen human proteins that could interact with 3A protein of EV71. Human β3 subunit of Na+/K+-ATPase (ATP1B3) protein was demonstrated to interact with the 3A protein of EV71. Although 3A protein had no effect on the expression of ATP1B3, EV71 infection resulted in elevated expression of ATP1B3 in RD cell line, both on messenger RNA (mRNA) and protein levels. Interestingly, knockdown of ATP1B3 could significantly increase the replication of EV71, whereas overexpression of ATP1B3 significantly suppressed the replication of EV71 in RD cells. Furthermore, we demonstrated that the expression of ATP1B3 could induce the production of type-I interferons. Our study demonstrated that ATP1B3 inhibit EV71 replication by enhancing the production of type-I interferons, which could act as a potential therapeutic target in EV71 infection.

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

Reporter Mutation Studies Show That Nicotinic Acetylcholine Receptor (nAChR) α5 Subunits and/or Variants Modulate Function of α6*-nAChR

To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/β3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human β3 subunit) almost completely abolishes the very small amount of function seen for α6β4*-nAChR and does not induce function of α6β2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6β4*-nAChR or induce function of α6β2*-nAChR. However, coexpression with mutant chimeric α5/β3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6β4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/β3(V9)'(S) subunits to have a gain-of-function effect on α6β2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either β2 or β4 subunits have some function that is modulated in the presence of α5 or α5/β3 subunits. Coexpression with α5/β3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.

Murine Platelets Expressing a Polymorphism of Integrin β3 Associated with Acute Coronary Thrombosis in Humans.

Inheritance of a naturally occurring polymorphism of the human integrin β3-subunit at amino acid 33 causes a change from Leu to Pro that has been implicated as a risk factor for thrombotic complication in humans. It is unclear; however, if Pro (also known as the platelet alloantigen PlA2 form of β3) can alter integrin activation to significantly affect platelet function in vivo, since some clinical studies do not corroborate the initial findings. Since PlA2 is a frequent variant of β3 expressed on 28% of platelets from Americans the impact of this genetic variation on platelet function could have widespread clinical relevance; therefore, we developed a murine model to determine if expression of this common polymorphism of human integrin β3 can increase platelet function and lead to an increased propensity for thrombosis within a homogeneous population of mice. This model eliminates issues arising from variation in the genetic make-up, physical environment, and/or lifestyles of humans and allows for in vivo analysis not possible with human subjects. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector under the transcriptional control of the human αIIb gene promoter to direct synthesis of β3 specifically to the megakaryocyte lineage. Bone marrow was isolated from β3-deficient mice and transduced with β3 virions encoding either the PlA1 or PlA2 form of human β3, and then transplanted into lethally irradiated β3-deficient littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of circulating platelets. Immunoanalysis using monoclonal antibodies and human serum that react specifically with the PlA1 or PlA2 confirmed the identity of each alloantigen of human β3. The lentivirus contained a second transgene encoding a drug-selectable marker (P140K MGMT) that was used for in vivo enrichment of transduced cells in mice treated with two regimens of cytotoxic reagents, O6-BG/BCNU. Flow cytometric analysis showed that use of drug-selection resulted in increased expression of the integrin αIIbβ3 complex to equal levels on nearly 100% of platelets using either form of human β3. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates ex vivo upon treatment with a cocktail of physiological agonists of platelet activation (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide).These results demonstrate the feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine and compare the effect of integrin αIIbβ3 structure on platelet function and thrombosis in vivo.

920. Assessing the Risk from Transfer of an Integrin β3 Gene That Encodes a Naturally Occurring Polymorphism Associated with Acute Coronary Thrombosis

Glanzmann thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder that is characterized by genetic defects of the platelet-specific integrin, αIIbβ3. Molecular defects in either the αIIb- or β3-subunit can disrupt receptor synthesis, assembly, and/or function thereby preventing platelets from binding the receptors major adhesive ligands (von Willebrand factor and fibrinogen) to form platelet aggregates as a primary response to vascular injury. When planning genetic transfer strategies for hematological disorders, it may be important to consider thrombotic risk factors associated with some naturally occurring polymorphisms of the transgene before choosing a cDNA cassette for gene therapy. This is exemplified in the debate concerning the increased risk of acute coronary thrombosis in association with inheritance of the platelet alloantigen (PlA2) form of the integrin β3 subunit encoding a proline at amino acid 33 versus PlA1 that has a leucine at residue 33. As a model to examine the suitability for expressing alternate PlA forms of β3 as gene therapy for GT, hematopoietic stem cells were transduced with either form of β3 (PlA1, PlA2 or PlA1/2) and then transplanted into a homogenous population of mice deficient in β3. To accomplish this, cDNA encoding each form of the human β3-subunit was subcloned into an HIV type-1 lentivirus-derived vector. The 3'-LTR viral enhancer/promoter was absent from this construct to produce a self-inactivating vector and the human αIIb gene promoter (nucleotides -889 to +35) was used to direct megakaryocyte-specific synthesis of β3. The vector utilized the woodchuck hepatitis virus postregulatory element, and the central polypurine tract to enhance the efficiency of transgene expression. Mononuclear cells isolated from bone marrow of β3 deficient mice were prestimulated for 2 days in medium containing cytokines, transduced with β3 virions and transplanted into lethally-irradiated littermates. Flow cytometric analysis demonstrated stable expression of the hybrid murine/human αIIbβ3 integrin complex on the surface of 30–50% circulating platelets from 2 weeks post-transplant. Immuno-analysis also indicated that expression of β3 was confined exclusively in platelets. Cytometric analysis using monoclonal antibodies and human serum that reacts specifically with the PlA1 or PlA2 were used to confirm expression of each subunit. Similar to platelets from normal mice, platelets expressing each PlA form of β3 could be induced to form aggregates in vitro upon stimulation with a cocktail of physiological platelet activation agonists (adenosine diphosphate, epinephrine and the thrombin receptor activating peptide). In contrast, platelets isolated from β3 deficient mice were unable to aggregate upon stimulation with physiological agonists. The results from this study demonstrate feasibility for targeting expression of altered forms of the human integrin β3-subunit to murine platelets and pave the way for future studies to examine the effect of these differences on platelet function.

High-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit.

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.

Enzymatic Properties of Human Na,K-ATPase α1β3Isozyme

Recent results of a wide-scale human cDNA sequencing project have identified a cDNA which encodes a hitherto unknown human protein sequence exhibiting structural similarities with β-subunits of the Na,K- and H,K-ATPase family and with the amphibian Na,KATPase β3-subunit, in particular. In this study the ability of the putative human β3-subunit to assemble with the human α1-subunit in functionally active Na,KATPase was examined using the baculovirus expression system. The recombinant baculovirus simultaneously expressing both α1and β3human proteins was produced using the dual-promoter transfer vector p2Bac. The expression of both human proteins in baculovirus-infected Sf-9 cell membranes detected with specific antibodies resulted in the formation of a catalytically competent α1β3ATPase complex. Characterization of the recombinant ATPase complex involved the analysis of Na+, K+, and ATP dependencies of enzyme activity and its sensitivity toward ouabain. Preparations of HeLa cell membranes containing α1β1isozyme of human Na,K-ATPase were used as control. The data obtained clearly demonstrated that α1β3ATPase exhibits enzymatic properties which are characteristic of Na,K-ATPase. The recombinant α1β3isozyme displayed significantly lower sensitivity to ouabain than native α1β1. These findings indicate that the hitherto unknown α1β3isozyme of human Na,K-ATPase is likely to existin vivo,thus suggesting further expansion of human Na,K-ATPase isozyme diversity. The present studies are the first in which heterologous expression has been used for the characterization of an isozyme of human Na,K-ATPase.

Genomic Organization of Human β3 Subunit Gene of Voltage-Gated Calcium Channel

Genomic Organization of Human β3 Subunit Gene of Voltage-Gated Calcium Channel

Cloning and sequencing of a rat heart cDNA encoding a G-protein β subunit related to the human retinal β3 subunit

A cDNA ( pJ5) encoding a protein with significant sequence identity to the previously known β1, β2, β3 and β4 subunits of guanine-nucleotide-binding proteins (G proteins) has been cloned from a 8–12-week-old rat heart cDNA library. Its predicted amino acid (aa) sequence of 340 aa most closely resembles that of the human β3 subunit, showing 96% identity. Northern blot analysis revealed that the major mRNA corresponding to the rat pJ5 cDNA is 2.0 kb in length. It is expressed at a high level in the heart and at a much lower level in the brain.

The GABA A receptor β3 subunit gene: Characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7

A cDNA encoding the human GABA A receptor β3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human β3 subunit amino acid sequence with β3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse β3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.