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K Geering

Publications and source records attributed to K Geering.

At least 19 recordsLinked to original sources
PubMed1992

Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.

Animals↗
PubMed1992

Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase.

Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.

Animals↗
PubMed1992

Processing, intracellular transport, and functional expression of endogenous and exogenous alpha-beta 3 Na,K-ATPase complexes in Xenopus oocytes.

The minimal functional Na,K-ATPase unit is composed of a catalytic alpha-subunit and a glycosylated beta-subunit. So far three putative beta-isoforms have been described, but only beta 1-isoforms have been identified clearly as part of a purified active enzyme complex. In this study we provide evidence that a putative beta 3-isoform might be the functional component of Xenopus oocyte Na,K-ATPase. beta 3-isoforms are expressed in the oocyte plasma membrane together with alpha-subunits, but beta 3-isoforms are synthesized to a lesser extent than alpha-subunits. The unassembled oocyte alpha-subunits accumulate in an immature trypsin-sensitive form most likely in the endoplasmic reticulum (ER). Injection of both beta 1- and beta 3-cRNA into oocytes abolishes the transport constraint of the oocyte alpha-subunit, renders it trypsin-resistant, and finally leads to an increased number of functional pumps at the plasma membrane. In addition, beta 3-isoforms as beta 1-isoforms depend on the concomitant synthesis of alpha-subunits to be able to leave the ER and to become fully glycosylated. Finally, alpha-beta 1 and alpha-beta 3 complexes expressed at the plasma membrane appear to have similar transport properties as assessed by ouabain binding, rubidium uptake, and electrophysiological measurements in oocytes coexpressing exogenous alpha 1- and beta 1- or beta 3-isoforms. Thus our data indicate that beta 3-isoforms have functional qualities similar to beta 1-isoforms. They can assemble and impose a structural reorganization to newly synthesized alpha-subunits which permits the exit from the ER and the expression of functional Na,K-pumps at the plasma membrane.

Animals↗
PubMed1992

Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes.

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.

Animals↗
PubMed1992

Subunit assembly and posttranslational processing of Na(+)-pumps.

Assembly, posttranslational processing and intracellular transport of Na,K-ATPase subunits are closely linked processes. Only coexpression of alpha- and beta-subunits in Xenopus oocytes permits the structural maturation and the ER exit of the two subunits. A putative beta 3-isoform of Na,K-ATPase and the beta-subunit of H,K-ATPase can support similar functions to those of beta 1-subunits of Na,K-ATPase in allowing the functional expression of alpha-beta complexes with Na,K-pump transport properties in the plasma membrane. These data indicate that a primary role of the beta subunit is to impose a conformational change on the newly synthesized alpha-subunits probably involved in the acquisition of transport competence and functional maturity of the catalytic alpha-subunit.

Animals↗
PubMed1991

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.

Adenosine Triphosphatases↗
PubMed1991

Coexpression of alpha 1 with putative beta 3 subunits results in functional Na+/K+ pumps in Xenopus oocytes.

The active Na+/K+ pump is composed of an alpha and a beta subunit. Until now, three putative isoforms of the beta subunit have been identified that share sequence similarity. We have expressed the beta 1 and beta 3 isoforms of Xenopus laevis Na+/K(+)-ATPase in Xenopus oocytes to compare functional properties of the Na+/K+ pump, including either of these two isoforms. Na+/K+ pump current, estimated as K(+)-induced outward current in voltage-clamped oocytes, was doubled by coexpression of alpha 1 subunits with either isoform of the beta subunit compared to expression of alpha 1 subunits alone. The kinetics of activation by external K+ and the voltage dependence of the electrogenic activity of the Na+/K+ pump were similar with both beta isoforms, indicating that both beta 1 and beta 3 isoforms can support expression at the oocyte surface of an active Na+/K+ pump with similar functional properties.

Animals↗
PubMed1991

Functional expression of N-terminal truncated alpha-subunits of Na,K-ATPase in Xenopus laevis oocytes.

N-terminal deletion mutants of Na,K-ATPase alpha 1 isoforms initiating translation at Met34 (alpha 1T1) or at Met43 (alpha 1T2) were expressed in X. laevis oocytes. Compared to beta 3 cRNA injected controls, the co-expression of alpha 1wt, alpha 1T1, alpha 1T2 with beta 3 subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K1/2 for potassium activation of the alpha 1T2/beta 3 Na,K-pumps is significantly higher than that of the alpha 1wt/beta 3 or alpha 1T1/beta 3 Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.

Amino Acid Sequence↗
PubMed1991

The functional role of the beta-subunit in the maturation and intracellular transport of Na,K-ATPase.

The minimal functional enzyme unit of Na,K-ATPase consists of an alpha-beta complex. The alpha-subunit bears all functional domains of the enzyme and so far a regulatory role for the beta-subunit in the catalytic cycle has not been established. On the other hand, increasing experimental evidence suggests that the beta-subunit is an indispensable element for the structural and functional maturation of the enzyme as well as its intracellular transport to the plasma membrane. This brief review summarizes the experimental data supporting the hypothesis that assembly of the beta-subunit is needed for the alpha-subunit to acquire the correct, stable configuration necessary for the acquisition of functional properties and its exit from the ER.

Animals↗
PubMed1991

Modulation of Na,K-ATPase expression during early development of Xenopus laevis.

In amphibian and mammalian systems, regulation of Na+ transport via the Na,K-ATPase plays an important role in distinct developmental processes such as blastocoele formation and neurulation. In this study, we have followed the Na,K-ATPase activity, the biosynthesis, and the cellular accumulation of catalytic alpha-subunits after fertilization of Xenopus laevis eggs up to neurula formation. Our data show that Na,K-ATPase activity increases significantly between stages 4 and 6 and again between stages 13 and 24. The four-fold rise in Na,K-ATPase activity during blastocoele formation is not mediated by an increased cellular pool of alpha-subunits. On the other hand, a five-fold increase of the biosynthesis rate around midblastula precedes a progressive accumulation up to neurula stage mainly of alpha 1-subunits and to a lesser extent of a second alpha-immunoreactive species. In contrast, newly synthesized glycoproteinic beta 1-subunits of Na,K-ATPase cannot be detected up to late neurula. These data indicate that (1) upregulation of Na,K-ATPase activity during blastocoele and neurula formation are mediated by different regulation mechanisms and (2) alpha- and possibly beta-isoforms are expressed in a developmentally regulated fashion during early Xenopus development.

Animals↗
PubMed1990

Mutual dependence of Na,K-ATPase alpha- and beta-subunits for correct posttranslational processing and intracellular transport.

In this study, we have followed the fate of newly synthesized alpha- and beta-subunits of Na,K-ATPase in Xenopus oocytes injected with alpha and/or beta cRNA to examine whether assembly of the two subunits is needed for a correct folding and/or for intracellular transport of Na,K-ATPase. Our data indicate that (1) assembly of alpha- and beta-subunits occurs at the level of the ER, (2) beta-subunits are needed for the newly synthesized alpha-subunit to adopt a stable configuration and (3) alpha- and beta-subunits mutually depend on each other to be transported out of the ER.

Animals↗
PubMed1990

The sodium pump needs its beta subunit.

The sodium pump Na,K-ATPase, located in the plasma membrane of all animal cells, is a member of a family of ion-translocating ATPases that share highly homologous catalytic subunits. In this family, only Na,K-ATPase has been established to be a heterodimer of catalytic (alpha) and glycoprotein (beta) subunits. The beta subunit has not been associated with the pump's transport or enzymatic activity, and its role in Na,K-ATPase function has been, until recently, a puzzle. In this review we describe what is known about the structure of beta and summarize evidence that expression of both alpha and beta subunits is required for Na,K-ATPase activity, that inhibition of glycosylation causes a decrease in accumulation of both alpha and beta subunits, and we provide evidence that pretranslational up-regulation of beta alone can lead to increased abundance of sodium pumps. These findings are all consistent with the hypothesis that the beta subunit regulates, through assembly of alpha beta heterodimers, the number of sodium pumps transported to the plasma membrane.

Animals↗
PubMed1989

Cross-reactivity of an antiserum to the alpha-subunit of the Na+, K+-ATPase of toad (Bufo marinus) kidney with basal and apical membranes of transporting epithelia of the rat.

An antibody to the 96 kD alpha-subunit of the Na+, K+-ATPase from Bufo marinus has been used in immunostaining rat kidney and salivary glands. Intense staining was observed on basolateral membranes of distal tubules of the kidney and striated ducts of the three major salivary glands. Less intense staining was seen on the basolateral membranes of parotid acinar cells, but no staining was seen on the acinar cells of submandibular or sublingual glands. These sites of staining have been shown, by other methods, to posses substantial Na+, K+-ATPase, indicating that the antibody recognizes antigenic determinants of the sodium pump highly conserved in the course of evolution. In addition, staining with this antibody was observed at the apical region of cells of the proximal straight tubule and of the papillary collecting duct in the kidney. Absorption studies suggest that the apical antigenic determinants are the same or closely related to each other but are distinct from basolateral antigenic determinants.

Animals↗
PubMed1989

Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.

Amiloride↗
PubMed1989

Inhibition of N-glycosylation affects transepithelial Na+ but not Na+-K+-ATPase transport.

Tunicamycin (TM) was used in toad urinary bladder (TBM) cells to study the role of N-glycosylation of the beta-subunit of Na+-K+-ATPase. Inhibition of the beta-subunit core glycosylation was dose dependent and coincided with a specific 70% decrease in newly synthesized beta- and alpha-subunits. Na+-K+-ATPase activity paralleled the decrease in the cellular content of the alpha-subunit, although the cellular and cell surface-expressed Na+-K+-ATPase pool was progressively filled up with nonglycosylated beta-subunits. In addition, the decrease in maximal Na+ transport capacity of the Na+-K+-ATPase as assessed by short-circuit current (SCC) measurements in the presence of amphotericin B correlated with the decrease in the total cell surface-expressed beta-subunit population despite the fact that it was composed of 47% nonglycosylated beta-subunits after 42 h of TM treatment. These results are consistent with the interpretation that beta-subunit glycosylation is not important either for the enzyme's intracellular sorting to the plasma membrane or its hydrolytic and transport properties. Finally, TM produced effects on basal SCC and electrical resistance that differed in their times of onset and time periods needed for recovery. Thus, in addition to the Na+-K+-ATPase, other glycoproteins in the apical membrane and the tight junctions must be implicated in the maintenance of transepithelial Na+ transport.

Adenosine Triphosphate↗
PubMed1989

A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes.

In all cellular systems studied so far, the catalytic alpha- and the glycosylated beta-subunit of Na+-K+-ATPase are coordinately synthesized and are assembled into stoichiometric alpha, beta-complexes. In contrast to these data, in this study we show that the fully grown oocyte of Xenopus laevis synthesizes much less beta-subunit than alpha-subunit. The alpha-subunit produced in excess over the beta-subunit is membrane associated but highly trypsin sensitive and can be compared with the immature alpha-subunit population identified in epithelial cells immediately after synthesis (K. Geering, J. P. Kraehenbuhl, and B.C. Rossier, J. Cell Biol. 105: 2613-2619, 1987). The Xenopus oocyte thus turns out to be a unique system to study the functional role of the beta-subunit. Injection of beta-subunit-specific mRNA transcribed in vitro from a beta-cDNA clone (derived from Xenopus kidney, A6 cells) into oocytes results in translation of a glycosylated beta-subunit. The synthesis of this exogenous beta-subunit increases significantly the proportion of trypsin-resistant oocyte alpha-subunits able to perform cation-dependent conformational changes. In addition, 25-65% more ouabian binding sites are expressed at the plasma membrane in beta-mRNA-injected oocytes. In contrast, newly synthesized alpha-subunit translated after injection of size-fractionated mRNA enriched in alpha-mRNA remains trypsin sensitive as the oocyte alpha-subunit. These data suggest that association of the beta-subunit to the alpha-subunit provokes a structural rearrangement of the alpha-subunit that might be a first step toward the functional maturation of the Na+-K+-ATPase and its expression at the plasma membrane.

Animals↗