Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.     

Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.     

Assembly of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase with its pre-existing beta-subunit in Xenopus oocytes

The alpha- and beta-subunits of Torpedo californica Na+/K(+)-ATPase were expressed in turn in single oocytes by alternately microinjecting the specific mRNAs for the alpha- and beta-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing beta-subunit, which had been synthesized by injecting mRNA for the beta-subunit, could assemble with the alpha-subunit expressed later in the single oocytes and the resulting alpha beta complex acquired both ouabain-binding and Na+/K(+)-ATPase activities. On the other hand, formation of the alpha beta complex was not detected when the alpha-subunit was expressed first, followed by the beta-subunit. These data suggest that the beta-subunit acts as a receptor or a stabilizer for the alpha-subunit upon the biogenesis of Na+/K(+)-ATPase.     

A possible role of the beta-subunit of (Na,K)-ATPase in facilitating correct assembly of the alpha-subunit into the membrane

mRNAs from the alpha- and beta-subunits (mRNA alpha and mRNA beta, respectively) of Torpedo californica (Na,K)-ATPase were injected into Xenopus laevis oocytes either separately or in combination, and the properties of the two subunits synthesized were studied. The alpha-subunit synthesized in oocytes injected with mRNA alpha alone was recovered in both the membrane and cytosol fractions and was susceptible to tryptic attack. When mRNA beta was coinjected with mRNA alpha, almost all the alpha-subunit was found in the membrane fraction and was resistant to trypsin. In all cases, essentially all of the beta-subunit was recovered in the membrane fraction and was resistant to trypsin. As the amount of mRNA beta coinjected increased, the amounts of both the alpha- and beta-subunits as well as (Na,K)-ATPase activity of the membrane fraction increased. These results suggest that the beta-subunit facilitates the correct assembly of the alpha-subunit into the membrane probably by forming a stable complex with the nascent alpha-subunit.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine chorionic gonadotropin beta-subunit

The complete amino acid sequence of the beta-subunit of equine chorionic gonadotropin (eCG beta) has been established by both automated Edman and manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradations. Specific fragments were produced by cleavage with Staphylococcus aureus V8 protease, trypsin, or dilute HCl. For the sequence analyses of the heavily glycosylated COOH-terminal portion, a chemical deglycosylation procedure with trifluoromethanesulfonic acid was employed. The peptide chain of eCG beta consists of 149 amino acid residues. Five or more oligosaccharide chains are attached to the protein, 1 unit linked by an N-glycosidic bond to asparagine at residue 13 and four or more units linked by O-glycosidic bonds to serine or threonine at residues in the COOH-terminal portion. The carbohydrate-bearing hydroxy amino acids have not yet been rigorously established. As compared to the beta-subunits of the pituitary gonadotropin hormones, lutropin, follitropin, and thyrotropin, eCG beta possesses a glycosylated COOH-terminal extension of about 30 amino acid residues, as does the human chorionic gonadotropin beta-subunit (hCG beta). When the comparison is restricted inside the disulfide bond-containing core (residues 1-110), the beta-subunit of eCG is highly homologous to hCG beta (66%). On the other hand, although the overall structural features closely resemble each other, much less homology exists in the COOH-terminal extensions of eCG beta and hCG beta.     

Hydrophobic interactions mediate binding of the glycine receptor beta-subunit to gephyrin

Glycine receptors (GlyRs) are ligand-gated chloride channel proteins composed of alpha- and beta-subunits. GlyRs are located to and anchored at postsynaptic sites by the receptor-associated protein gephyrin. Previous work from our laboratory has identified a core motif for gephyrin binding in the cytoplasmic loop of the GlyR beta-subunit. Here, we localized amino acid residues implicated in gephyrin binding by site-directed mutagenesis. In a novel transfection assay, a green fluorescent protein-gephyrin binding motif fusion protein was used to monitor the consequences of amino acid substitutions for beta-subunit interaction with gephyrin. Only multiple, but not single, replacements of hydrophobic side chains abolished the interaction between the two proteins. Our data are consistent with gephyrin binding being mediated by the hydrophobic side of an imperfect amphipathic helix.     

Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase. In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate

The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z. (1985) FEBS Lett. 178, 10-14). Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E. coli F1-ATPase. Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supported assembly and TNP-ADP did not. The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.     

Identification of a novel gene of the X,K-ATPase beta-subunit family that is predominantly expressed in skeletal and heart muscles.

We have identified the fifth member of the mammalian X,K-ATPase beta-subunit gene family. The human and rat genes are largely expressed in skeletal muscle and at a lower level in heart. The deduced human and rat proteins designated as beta(muscle) (beta(m)) consist of 357 and 356 amino acid residues, respectively, and exhibit 89% identity. The sequence homology of beta(m) proteins with known Na,K- and H,K-ATPase beta-subunits are 30.5-39.4%. Unlike other beta-subunits, putative beta(m) proteins have large N-terminal cytoplasmic domains containing long Glu-rich sequences. The data obtained indicate the existence of hitherto unknown X,K-ATPase (most probably Na,K-ATPase) isozymes in muscle cells.     

Tryptic activation of the insulin receptor. Proteolytic truncation of the alpha-subunit releases the beta-subunit from inhibitory control

Trypsin exerts insulin-like effects in intact cells and on partially purified preparations of insulin receptors. To elucidate the mechanism of these insulinomimetic effects, we compared the structures of insulin- and trypsin-activated receptor species with their functions, including insulin binding, autophosphorylation, and tyrosine kinase activity. In vitro treatment of wheat germ agglutinin-purified receptor preparations with trypsin resulted in proteolysis of both alpha- and beta-subunits. The activated form of the receptor had an apparent molecular mass of 110 kDa under nonreducing conditions, compared to the 400-kDa intact receptor, and was separated following reduction into an 85-kDa beta-subunit related fragment and a 25-kDa alpha-subunit related fragment. Treatment of whole cells with trypsin prior to isolation of the insulin receptor resulted in proteolytic modification of the alpha-subunit only. In this case, the total molecular mass of the activated species was 116 kDa, comprised of an intact 92-kDa beta-subunit and again a 25-kDa alpha-subunit related fragment. Values of Km for peptide substrate phosphorylation and Ki for inhibition of receptor autophosphorylation, and sites of autophosphorylation within the beta-subunits were similar for receptors activated either by insulin or trypsin. Insulin had no additional effect on the rate of autophosphorylation of the truncated receptor, and no binding of insulin by the truncated receptor was detected either by direct assay or cross-linking with bifunctional reagents. Based on the deduced amino acid sequence of the insulin receptor and the structural studies presented here we concluded that this activated form of the receptor resulted from tryptic cleavage at the dibasic site Arg576-Arg577. This was accompanied by loss of the insulin binding site and separation of alpha-beta heterodimers. As truncation of the alpha-subunit results in beta-subunit activation, it appears that the beta-subunit is a constitutively activated kinase and that the function of the alpha-subunit in the intact receptor is to inhibit the beta-subunit.     

Terminal deoxynucleotidyltransferase. Alignment of alpha- and beta-subunits of the core enzyme along the primary translation product.

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

Expression, localization, and function of an N-terminal half fragment of the rat Na,K-ATPase beta-subunit in HeLa cells.

The N-terminal half of the beta-subunit of rat brain Na,K-ATPase was expressed in HeLa cells transfected with the plasmid pSV2TKneo beta N containing the truncated beta-subunit cDNA to study the assembly and transport of alpha-beta complex. Immunoprecipitation from extracts of metabolically labeled transformed cells demonstrated that the truncated beta-subunit polypeptide (beta N) was neither transported to the plasma membrane nor assembled into an alpha-beta complex with the endogenous alpha-subunit. Cell fractionation experiments showed that the beta N truncated subunit remained unassembled within rough microsomes, suggesting that it never exited from the endoplasmic reticulum (ER). The assembly of the endogenous alpha-and beta-subunits in the beta N-expressing cells was significantly inhibited compared with control cells or with the transformants that did not express the beta N. These results suggest that the N-terminal portion of the beta-subunit interferes with the normal assembly of the endogenous complex which normally takes place in the ER.     

Cytoplasmic and transmembrane domain deletions of Na,K-ATPase beta-subunit. Effects on subunit assembly and intracellular transport.

cDNAs encoding Na,K-ATPase beta-subunits containing deletions in the cytoplasmic domain or in the single membrane-spanning domain of the molecule were constructed and expressed in mouse L cells to determine the effect(s) of deletions in these domains on alpha/beta-subunit assembly and intracellular targeting. Avian beta-subunits lacking some or all of the cytoplasmic domain (endodomain) assemble with the endogenous mouse alpha-subunit and are correctly transported to the plasma membrane. Mutants containing deletions in the transmembrane domain were constructed by fusing portions of cDNAs encoding the amino-terminal one-third of human beta-subunit deletion mutants with avian beta-subunit cDNA encoding the carboxyl two-thirds of the molecule. A deletion of 3 amino acids in transmembrane domain resulted in correct alpha/beta-subunit assembly and localization to the plasma membrane. In contrast, deletions of 5 or more amino acids in the transmembrane domain prevented expression of the beta-subunit at the cell surface and resulted in the accumulation of these molecules in the ER. In spite of these targeting differences, all beta-subunit mutants capable of membrane insertion were also able to assemble with the alpha-subunit. These results suggest that the specificity for alpha/beta assembly resides in the ectodomains of the subunits.     

Role of the beta-subunit arginine/lysine finger in integrin heterodimer formation and function

Formation of the integrin alphabeta heterodimer is essential for cell surface expression and function. At the core of the alphabeta interface is a conserved Arg/Lys "finger" from the beta-subunit that inserts into a cup-like "cage" formed of two layers of aromatic residues in the alpha-subunit. We evaluated the role of this residue in heterodimer formation in an alphaA-lacking and an alphaA-containing integrin alphaVbeta3 and alphaMbeta2 (CD11b/CD18), respectively. Arg261 of beta3 was mutated to Ala or Glu; the corresponding Lys252 of beta2 was mutated to Ala, Arg, Glu, Asp, or Phe; and the effects on heterodimer formation in each integrin examined by ELISA and immunoprecipitation in HEK 293 cells cotransfected with plasmids encoding the alpha- and beta-subunits. The Arg261Glu (but not Arg261Ala) substitution significantly impaired cell surface expression and heterodimer formation of alphaVbeta3. Although Lys252Arg, and to a lesser extent Lys252Ala, were well tolerated, each of the remaining substitutions markedly reduced cell surface expression and heterodimer formation of CD11b/CD18. Lys252Arg and Lys252Ala integrin heterodimers displayed a significant increase in binding to the physiologic ligand iC3b. These data demonstrate an important role of the Arg/Lys finger in formation of a stable integrin heterodimer, and suggest that subtle changes at this residue affect the activation state of the integrin.     

Insulin receptors in rat brain: insulin stimulates phosphorylation of its receptor beta-subunit

In rat brain cortex synaptosomes insulin stimulated the phosphorylation of its own receptor beta-subunit (94 kDa) as identified by immunoprecipitation with anti-insulin or anti-receptor antiserum. The receptor alpha-subunit (115 kDa) was characterized by specific labeling with 125I-labeled photoreactive insulin. These observations indicate that: (i) insulin receptors in brain are composed of alpha-subunits which bind insulin, and beta-subunits, the phosphorylation of which can be stimulated by insulin; (ii) the size of alpha-subunits in brain is significantly smaller than in other tissues (115 vs 130 kDa), whereas beta-subunits (94 kDa) are identical. We suggest that brain insulin receptors represent a subtype regarding their binding function, whereas their enzyme function is more conserved.     

Phosphorylation and activation of the cardiac isoenzyme of phosphorylase kinase by the cAMP-dependent protein kinase

The cAMP-dependent protein kinase catalyzes the phosphorylation of the alpha- and beta-subunits of the cardiac isozyme of phosphorylase kinase. beta-Subunit phosphorylation achieves a maximum level of between 1 to 2 mol of phosphate/mol of phosphorylase kinase, a value less than the stoichiometric content of beta-subunits in the enzyme. This, less than stoichiometric incorporation, is not a result of the presence of endogenous phosphate in equivalent sites in the remaining beta-subunit moieties. Pretreatment of phosphorylase kinase with phosphoprotein phosphatase, under conditions proven to dephosphorylate such sites, does not modify the observed extent of beta-subunit phosphorylation. alpha'-Subunit phosphorylation is initiated at a slower rate than beta but achieves a higher maximum level of incorporation. alpha'-Subunit phosphorylation, but not the extent of beta-subunit phosphorylation, is stimulated by MnCl2 and partially inhibited by NaF; neither is effected by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The activation of cardiac phosphorylase kinase that occurs concomitantly with phosphorylation appears to be dependent upon phosphate incorporation into both the alpha- and beta-subunits. At low levels of activation a close correlation is observed between activation and either alpha-subunit phosphorylation, beta-subunit phosphorylation, or total phosphorylation. However, the cAMP-dependent catalyzed phosphorylation of alpha, at a time after which beta-subunit phosphorylation is already maximal, also results in activation of cardiac phosphorylase kinase.     

Pig kidney Na+,K+-ATPase. Primary structure and spatial organization

cDNAs complementary to pig kidney mRNAs coding for alpha- and beta-subunits of Na+,K+-ATPase were cloned and sequenced. Selective tryptic hydrolysis of the alpha-subunit within the membrane-bound enzyme and tryptic hydrolysis of the immobilized isolated beta-subunit were also performed. The mature alpha- and beta-subunits contain 1016 and 302 amino acid residues, respectively. Structural data on the peptides from extramembrane regions of the alpha-subunit and on glycopeptides of the beta-subunit underlie a model for the transmembrane arrangement of Na+,K+-ATPase polypeptide chains.     

The roles of carbohydrate chains of the beta-subunit on the functional expression of gastric H(+),K(+)-ATPase

Gastric H(+),K(+)-ATPase consists of alpha and beta-subunits. The alpha-subunit is the catalytic subunit, and the beta-subunit is a glycoprotein stabilizing the alpha/beta complex in the membrane as a functional enzyme. There are seven putative N-glycosylation sites on the beta-subunit. In this study, we examined the roles of the carbohydrate chains of the beta-subunit by expressing the alpha-subunit together with the beta-subunit in which one, several, or all of the asparagine residues in the N-glycosylation sites were replaced by glutamine. Removing any one of seven carbohydrate chains from the beta-subunit retained the H(+),K(+)-ATPase activity. The effects of a series of progressive removals of carbohydrate chains on the H(+),K(+)-ATPase activity were cumulative, and removal of all carbohydrate chains resulted in the complete loss of H(+), K(+)-ATPase activity. Removal of any single carbohydrate chain did not affect the alpha/beta assembly; however, little alpha/beta assembly was observed after removal of all the carbohydrate chains from the beta-subunit. In contrast, removal of three carbohydrate chains inhibited the surface delivery of the beta-subunit and the alpha-subunit assembled with the beta-subunit, indicating that the surface delivery mechanism is more dependent on the carbohydrate chains than the expression of the H(+),K(+)-ATPase activity and alpha/beta assembly.     

Chemical modification of tryptophan residues in Escherichia coli succinyl-CoA synthetase. Effect on structure and enzyme activity

Succinyl-CoA synthetase of Escherichia coli is an alpha 2 beta 2 protein containing active sites at the interfaces between alpha- and beta-subunits. The alpha-subunit contains a histidine residue that is phosphorylated during the reaction. The beta-subunit binds coenzyme A and probably succinate [see Nishimura, J. S. (1986) Adv. Enzymol. Relat. Areas Mol. Biol. 58, 141-172]. Chemical modification studies have been conducted in order to more clearly define functions of each subunit. Tryptophan residues of the enzyme were modified by treatment with N-bromosuccinimide at pH 7. There was a linear relationship between loss of enzyme activity and tryptophan modified. At one tryptophan residue modified per beta-subunit, 100% of the enzyme activity was lost. In this enzyme sample, one methionine residue in each alpha- and beta-subunit was oxidized to methionine sulfoxide, although loss of enzyme activity could not be related in a linear manner to the formation of this residue. Subunits were prepared from enzyme that was inactivated 50% by N-bromosuccinimide with 0.5 tryptophan modified per beta-subunit but with insignificant modification of methionine residues in either subunit. Small decreases in the tyrosine and histidine content were observed in the alpha-subunit but not in the beta-subunit. In this case, modified beta-subunit when mixed with unmodified alpha-subunit gave a population of molecules that was 50% as active as the refolded, unmodified control but was only slightly changed with respect to phosphorylation capacity and unchanged with respect to rate of phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)