Production and secretion in CHO cells of the extracellular domain of AMOGβ2, a type-II membrane protein

Gloor, Sergio M.; Nasse, Kira; Essen, Lars‐Oliver; Appel, F

doi:10.1016/0378-1119(92)90111-2

Cited by 12 publications

(12 citation statements)

References 11 publications

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“…This is as expected, because brain expresses both ␤ isoforms (Mercer et al, 1986;Pagliusi et al, 1989), whereas kidney expresses only or predominantly ␤1 (MartinVasallo et al, 1989). The mAb SM-GP50 reacted with the ␤2 truncated protein, confirming the report of Gloor et al (1992).…”

Section: ␤2supporting

confidence: 71%

Isoforms of Na,K-ATPase α and β Subunits in the Rat Cerebellum and in Granule Cell Cultures

1997

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There are multiple isoforms of the Na,K-ATPase in the nervous system, three isoforms of the ␣ subunit, and at least two of the ␤ subunit. The ␣ subunit is the catalytic subunit. The ␤ subunit has several roles. It is required for enzyme assembly, it has been implicated in neuron-glia adhesion, and the experimental exchange of ␤ subunit isoforms modifies enzyme kinetics, implying that it affects functional properties. Here we describe the specificities of antibodies against the Na,K-ATPase ␤ subunit isoforms ␤1 and ␤2. These antibodies, along with antibodies against the ␣ subunit isoforms, were used to stain sections of the rat cerebellum and cultures of cerebellar granule cells to ascertain expression and subcellular distribution in identifiable cells. Comparison of ␣ and ␤ isoform distribution with doublelabel staining demonstrated that there was no preferential association of particular ␣ subunits with particular ␤ subunits, nor was there an association with excitatory or inhibitory neurotransmission modes. Isoform composition differences were seen when Purkinje, basket, and granule cells were compared. Whether ␤1 and ␤2 are specific for neurons and glia, respectively, has been controversial, but expression of both ␤ subunit types was seen here in granule cells. In rat cerebellar astrocytes, in sections and in culture, ␣2 expression was prominent, yet the expression of either ␤ subunit was low in comparison. The complexity of Na,K-ATPase isoform distribution underscores the subtlety of its regulation and physiological role in excitable cells.

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Section: ␤2supporting

confidence: 71%

Isoforms of Na,K-ATPase α and β Subunits in the Rat Cerebellum and in Granule Cell Cultures

1997

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“…We therefore examined whether soluble ␤1 or ␤2 subunit ectodomains combine with one of the ␣ isoforms to form a detergent-resistant complex. Xenopus oocytes injected with appropriate cRNAs (24,25) secreted the fully glycosylated form of the mouse ␤1 and ␤2 ectodomains into the medium (not shown). Oocytes were then injected with cRNA for the ␤1 ectodomain together with cRNA for the Torpedo ␣1 subunit or for one of the rat ␣ isoforms.…”

Section: Resultsmentioning

confidence: 99%

“…cDNAs for the ␤1 subunit (21) and ␤2 subunit of the Na,K-pump of the mouse (22) in pGEM2 (Promega) have been described (23). cDNAs encoding ectodomains of the mouse ␤1 subunit (233 aa) and ␤2 subunit (228 aa) were described (24). Both cDNAs were subcloned into pSP64polyA (Promega).…”

Section: Methodsmentioning

confidence: 99%

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Schmalzing

Ruhl

Gloor

1997

Proc. Natl. Acad. Sci. U.S.A.

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The functional unit of the Na,K-ATPase consists of a catalytic ␣ subunit noncovalently linked with a glycoprotein subunit, ␤. Using ouabain binding assays and immunoprecipitation of rodent ␣͞␤ complexes, we show here that all six possible isozymes between three ␣ and two ␤ isoforms can be formed in Xenopus oocytes. Two isoformspecific differences in ␣͞␤ interactions are observed: (i) ␣1͞␤1 and ␣2͞␤2 complexes, in contrast to ␣1͞␤2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) ␤2 subunits must carry N-glycans to combine with ␣1 but not with ␣2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated ␤1 subunit comprising the ectodomain results in assembly with ␣1 and ␣2, but not with ␣3; the ␤2 ectodomain combines with ␣2 only. A chimera consisting of 81% and 19% of the ␣1 N terminus and ␣2 C terminus, respectively, behaves like ␣2 and coprecipitates with the ␤2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the ␤2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase ␣ and ␤ subunits.The Na,K-ATPase is a heterodimer consisting of a 110-kDa ␣ subunit that is noncovalently linked with a glycoprotein ␤ subunit (protein core, 33-35 kDa). The ␣ subunit is the catalytic active part and bears the site for ATP hydrolysis, the binding sites for Na ϩ , K ϩ , and cardiac glycosides (1). The ␣ subunit spans the membrane several times. The ␤ subunit exposes a short N-terminal tail to the cytoplasm and the majority of its mass at the extracellular surface. It confers conformational stability to the ␣ subunit during or after assembly in the endoplasmic reticulum and is needed for routing of the enzyme to the plasma membrane (2-5).Three ␣ isoforms (␣1, ␣2, and ␣3) and two ␤ isoforms (␤1 and ␤2) have been identified in mammals and birds, each encoded by a separate gene (6). The ␣ isoforms show more than 80% homology even for very diverse species. The two ␤ isoforms have only about 40% amino acid sequence identity within the same species. Sequences encoding a mammalian ␤3 isoform have been deposited in the GenBank database. Both ␣ and ␤ isoforms display complex patterns of tissue-specific and developmental expression (7,8).All six possible ␣͞␤ isozymes are formed from the three ␣ and two ␤ isoforms of birds (9) and mammals (this work) in vitro, supporting the assumption that different isozymes exist in vivo (10, 11). Hybrid Na,K-pumps are readily formed from ␣1 and ␤1 subunits of virtually all species investigated (for a review, see ref. 12). The ␤1 isoform can even be replaced by the ␤ subunit of the H,K-pump to yield a functional Na,Kpump (13).Protein domains involved in ␣1͞␤1 interactions have only partially been identified. Deletion of the cytoplasmic domain and part of the transmembrane domain of chicken ␤1 does not disturb assembly as long as the truncated mutants are inserted into the membrane (14). Chimeric ␤1 subunits containing the N terminus and the membrane spanning domain...

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“…There is a small hydrophobic region near the carboxyl terminus, making it at least possible that this protein crosses the membrane twice. However, a second membrane anchor can now be ruled out with certainty, since we have found that an N-terminally truncated mouse p2-subunit comprising the complete ectodomain is efficiently secreted into the external medium by both CHO cells [43] and Xenopus oocytes [unpubl. results].…”

Section: Structural Organization and Membrane Topology>mentioning

confidence: 99%

Na<sup>+</sup>/K<sup>+</sup>-Pump Beta Subunits: Structure and Functions

Schmalzing¹,

Gloor

1994

Cell Physiol Biochem

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This review summarizes evidence that Na⁺/K⁺-ATPase β-sub-units have two essential functions. First, β-subunits constitute an indispensable part of Na⁺/K⁺-ATPase, the minimum functional unit of which is an αβ-heterodimer, by stabilizing the correct folding of the catalytic α-sbunit. The various β-isoforms confer different K⁺ affinities to the mature enzyme. Second, β2-subunits are involved in cell-cell interactions. To account for both functions, we discuss the possibility that β-subunits can serve as recognition molecules which transmit extracellular information onto the α-subunit or by themselves interact with receptors of neighboring cells.

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Production and secretion in CHO cells of the extracellular domain of AMOGβ2, a type-II membrane protein

Cited by 12 publications

References 11 publications

Isoforms of Na,K-ATPase α and β Subunits in the Rat Cerebellum and in Granule Cell Cultures

Isoforms of Na,K-ATPase α and β Subunits in the Rat Cerebellum and in Granule Cell Cultures

Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

Na<sup>+</sup>/K<sup>+</sup>-Pump Beta Subunits: Structure and Functions

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