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M J Caplan

Publications and source records attributed to M J Caplan.

At least 37 records · Page 2Linked to original sources
PubMed1998

Gastric H+/K(+)-ATPase: targeting signals in the regulation of physiologic function.

The H+/K(+)-ATPase of gastric parietal cells catalyzes acid secretion in the stomach. Regulation of this pump's function involves its targeting to an intracellular storage compartment, which fuses with the apical plasma membrane in response to secretagogue stimulation. The H+/K(+)-ATPase must also be returned to this intracellular storage compartment to inactivate acid secretion. Recent research suggests that a number of targeting signals and protein-protein interactions participate in governing this process.

Animals↗
PubMed1998

A basolateral sorting signal is encoded in the alpha-subunit of Na-K-ATPase.

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the alpha-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric alpha-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase alpha-subunit contain a basolateral sorting signal. H-K-ATPase beta-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase beta-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase alpha-subunit masks or is dominant over any apical sorting information present in the beta-polypeptide. In gastric parietal cells, the H-K-ATPase beta-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase beta-subunit complex is directed to a similar compartment in LLC-PK1 cells, we treated transfected cells with drugs that raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase beta-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.

1-Methyl-3-isobutylxanthine↗
PubMed1998

A tyrosine-based signal regulates H-K-ATPase-mediated potassium reabsorption in the kidney.

Isoforms of the H-K-ATPase participate in active K resorption in the renal collecting tubule. The cytoplasmic tail of the beta-subunit of the gastric H-K-ATPase includes a 4 amino acid motif which is highly homologous to tyrosine-based endocytosis signals. We have generated transgenic mice expressing an H-K-ATPase beta-subunit in which the tyrosine residue in this sequence has been mutated to alanine. Mice expressing the mutated protein manifest constitutive hypersecretion of gastric acid, demonstrating that the beta-subunit tyrosine-based motif is required for the regulated endocytosis of the H-K pump and hence the cessation of gastric acid output. To test the possibility that the tyrosine-based sequence in the tail of the H-K-ATPase beta-subunit plays a role in regulating the function of renal H-K-ATPases, we examined renal K clearance in normal and in transgenic mice. Blood pressure, urine volume, glomerular filtration rate (GFR), plasma Na, and Na excretion are similar in control and transgenic mice. However, plasma K concentrations are significantly higher in transgenic mice (4.76 +/- 0.13 meq/l in transgenic and 4. 12 +/- 0.04 meq/l in control; n = 9, P < 0.05) and K excretion is lower in the transgenic animals (fractional excretion of K was 26.2 +/- 3.62% in transgenic and 50.1 +/- 4.78% in control; n = 9, P < 0. 01). These data suggest that the tyrosine-based signal in the cytoplasmic tail of the H-K-ATPase beta-subunit functions in the kidney as it does in the stomach to internalize H-K pump and thus inactivate pump function. Its elimination may result in the constitutive presence of the pump at the cell surface and lead to excessive urinary K reabsorption.

Animals↗
PubMed1998

Effects of okadaic acid, calyculin A, and PDBu on state of phosphorylation of rat renal Na+-K+-ATPase.

Several indirect lines of evidence suggest that protein kinases and phosphatases modulate the activity of renal Na+-K+-ATPase. The aim of this study was to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were prelabeled with [32P]orthophosphate and incubated with the inhibitors of protein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase alpha-subunit was evaluated by measuring the amount of [32P]phosphate incorporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [32P]phosphate incorporation into immunoprecipitated Na+-K+-ATPase alpha-subunit. OA and PDBu had a synergistic effect on the state of phosphorylation of Na+-K+-ATPase alpha-subunit. FK-506 did not affect Na+-K+-ATPase phosphorylation, neither alone nor in the presence of PDBu. Each of the drugs, OA, CL-A, and PDBu, inhibited the activity of Na+-K+-ATPase in microdissected proximal tubules. PDBu potentiated OA-induced inhibition of Na+-K+-ATPase activity. Inhibition of Na+-K+-ATPase required a lower dose of CL-A than of OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was approximately 1.5 nmol Pi. mg protein-1. min-1. Using a monoclonal anti-alpha antibody that fails to recognize the subunit when Ser23 is phosphorylated by PKC, we demonstrated that the dose response of PDBu inhibition of Na+-K+-ATPase correlated with the dose response of phosphorylation of the enzyme. The results suggest that the state of phosphorylation and activity of proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases.

Animals↗
PubMed1998

Conformational alterations resulting from mutations in cytoplasmic domains of the alpha subunit of the Na,K-ATPase.

This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between M4 and M5. In the first mutation (alpha 1M32), 32 residues were removed from the N-terminus. The second mutation (E233K) was in the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPase with the homologous segment (residues 356-519) of the gastric H,K-ATPase. The first two mutations, either separately or in combination (alpha 1M32E233K), shift the equilibrium between the major conformational states of the enzyme, E1 and E2, in favor of E1 as manifested by increased apparent affinity for ATP, lower catalytic turnover, and decreased sensitivity to inhibition by vanadate. The striking changes observed with alpha 1M32E233K suggests interactions between the N-terminus, the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one mutant involving substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is unaltered suggesting that changes in apparent affinity for ATP are secondary to changes in K+ ligation. The question of cation selectivity, in particular that of Na+ versus protons, has been addressed in structure/function analysis of a cytoplasmic chimera involving the M4-M5 loop. Transport studies performed in the presence or absence of Na+ and at low versus high pH indicate a marked alteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to cation binding in, presumably, the juxtapositioned transmembrane domain.

Amino Acid Substitution↗
PubMed1998

Sorting of P-type ATPases in polarized epithelial cells.

The Na,K-ATPase and the H,K-ATPase are highly homologous members of the P-type family of ion transporting ATPase. Despite their structural similarity, these two pumps are sorted to different destinations in polarized epithelial cells. While the Na,K-ATPase is restricted to the basolateral surfaces of most epithelial cells types, the H,K-ATPase is concentrated at the apical plasmalemma and in a pre-apical vesicular storage compartment in the parietal cells of the stomach. We have generated molecular chimeras composed of complementary portions of these two pumps' alpha-subunits. By expressing these pump constructs in polarized epithelial cells in culture, we have been able to identify sequence domains which participate in the targetting of the holoenzyme. We find that information embedded within the sequence of the fourth transmembrane domain of the H,K-ATPase is sufficient to account for this protein's apical localization. Stimulation of gastric acid secretion results in insertion of the intracellular H,K-ATPase pool into the apical plasma membrane and inactivation of acid secretion is accompanied by the re-internalization of these pumps. We have identified a tyrosine-based signal in the cytoplasmic tail of the H,K-ATPase beta-subunit which appears to be required for this endocytosis. We have mutated the critical tyrosine residue to alanine and expressed the altered protein in transgenic mice. The H,K-ATPase remains continuously at the apical cell surface in parietal cells from these animals, and they constitutively hypersecrete gastric acid. These results demonstrate that the beta-subunit sequence mediates the internalization of the H,K-ATPase and is required for the cessation of gastric acid secretion. Thus, at least two sorting signals are required to ensure the proper targetting and regulation of the gastric H,K pump.

Animals↗
PubMed1997

A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion.

Gastric acid secretion is mediated by the H/K-ATPase of parietal cells. Activation of acid secretion involves insertion of H/K-ATPase into the parietal cell plasmalemma, while its cessation is associated with reinternalization of the H/K-ATPase into an intracellular storage compartment. The cytoplasmic tail of the H/K-ATPase beta subunit includes a four residue sequence homologous to tyrosine-based endocytosis signals. We generated transgenic mice expressing H/K-ATPase beta subunit in which this motif's tyrosine residue is mutated to alanine. Gastric glands from animals expressing mutant beta subunit constitutively secrete acid and continuously express H/K-ATPase at their cell surfaces. Thus, the beta subunit's tyrosine-based signal is required for the internalization of H/K-ATPase and for the termination of acid secretion. As a consequence of chronic hyperacidity, the mice develop gastric ulcers and a hypertrophic gastropathy resembling Menetrier's disease.

Animals↗
PubMed1997

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells.

The gamma-aminobutyric acid transporter (GAT-1) isoform of the gamma-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DeltaC-GAT) maintained the original functional activity and apical localization, either the removal (DeltaC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.

Amino Acid Sequence↗
PubMed1997

The effect of premolar extractions on the soft-tissue profile in adult African American females.

The present study was designed to evaluate the effect of four first premolar extractions on the soft tissue profile in African American patients. Pretreatment and posttreatment cephalograms of 28 adult female patients were assessed. The data were subjected to ANOVA, and correlation coefficients were performed between the significantly different dental and soft tissue variables. Variables that showed correlation at r value of greater than 0.6 were subjected to a stepwise multiple regression. The results of the study indicate that retraction of the lower lip correlates with retraction of both maxillary and mandibular anterior teeth. A ratio of 1.2:1 [corrected] was obtained between mandibular incisor retraction and retraction of the lower lip. The relationship between the upper lip and retraction of maxillary incisors was not significant. A ratio of 1.75:1 [corrected] was attained between maxillary incisor retraction and upper lip change. The upper lip correlated most strongly with lower lip retraction. Mandibular incisor angulation was the only hard-tissue variable that could be used as a predictor in a regression model to explain lip response to orthodontic therapy. Changes in the maxillary complex were more difficult to predict because of the complex nature of the soft-tissue integument and the details of muscle tension and soft-tissue tone that were lost by conversion of a three-dimensional structure into a roentgenographic cephalogram. A significant profile change did occur following the extraction of four first premolars and subsequent orthodontic therapy.

Adolescent↗
PubMed1997

Sorting and trafficking of ion transport proteins in polarized epithelial cells.

Transport proteins are targeted to specific plasmalemmal domains in polarized epithelial cells. The molecular signals that govern these sorting events are just beginning to be elucidated. In many cases, the cell surface delivery of transport proteins is subjected to tight regulation. Several different mechanisms appear to participate in these trafficking processes.

Animals↗
PubMed1997

Ion pumps in epithelial cells: sorting, stabilization, and polarity.

The P-type family of ion-transporting ATPases includes all of the isoforms of Na(+)-K(+)-adenosinetriphosphatase (ATPase), the plasma membrane and organellar Ca(2+)-ATPases, gastric H(+)-K(+)-ATPase, and the recently characterized nongastric H(+)-K(+)-ATPases, among others. In epithelial cells, members of this pump family generate the cation gradients responsible for vectorial fluid and solute transport. To carry out this function, each P-type ATPase must be restricted to a specific membrane domain. Newly synthesized ion pumps must be sorted to their appropriate destinations and retained there after their delivery. Recently, progress has been made toward understanding the signals and targeting pathways that epithelial cells employ to generate anisotropic pump distributions. The mechanisms involved in generating these pump distributions may serve as well to regulate pump function.

Adenosine Triphosphatases↗
PubMed1997

Membrane polarity in epithelial cells: protein sorting and establishment of polarized domains.

Epithelial cells go to great trouble to organize the subdomains of their plasma membranes. The apical and basolateral surfaces of polarized epithelia are equipped with markedly distinct populations of channels, carriers, and pumps. This anisotropy is an absolute prerequisite for vectorial solute and fluid transport. The physiological properties of an individual epithelial cell type are determined not only by its census of transport proteins but also by the manner in which these proteins are segregated between the apical and basolateral portions of the plasmalemma (Curr. Top. Membr. 39: 37-86, 1991). To achieve this asymmetry, an epithelial cell must be able to establish distinct surface domains, to target newly synthesized transport proteins to their appropriate sites of functional residence, and to retain them there following their delivery. Studies of the cellular pathways involved in generating and maintaining the polarized state have begun to illuminate an elegant network of cell biological specializations that may be involved not only in establishing the distributions of transport proteins but in dynamically regulating their function as well.

Animals↗
PubMed1996

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.

Antibodies, Monoclonal↗
PubMed1996

Cell-specific sorting of biogenic amine transporters expressed in epithelial cells.

We have utilized polarized epithelial cells stably expressing neurotransmitter transporters to analyze the sorting behavior of these membrane proteins. The transporters for serotonin (5-HT), dopamine (DA), and norepinephrine (NE) are expected to be present in situ in the most distal extremities of axonal membranes, where they terminate the action of their biogenic amine substrates. Both Madin-Darby canine kidney (MDCK) and LLC-PK1 cells were stably transfected with cDNAs encoding either the rat 5-HT transporter (SERT), the human NE transporter (NET), or the rat or human DA transporter (DAT). These cells were grown on permeable filter supports, and the transporters were localized by three independent techniques. Confocal immunofluorescence microscopy indicated that each of the transporters expressed in LLC-PK1 cells was sorted to the basolateral membrane, co-localizing with the Na+/K+-ATPase. In MDCK cells, however, DAT was located primarily on the apical surface, while SERT and NET were found on the basolateral membranes. Cell surface biotinylation using an impermeant biotinylating reagent confirmed the immunocytochemistry results. Thus, SERT and NET in MDCK cells were labeled more efficiently from the basolateral medium than the apical medium, and DAT in MDCK cells was labeled more efficiently from the apical side than the basolateral side. Transport measurements in transfected MDCK cells agreed with the immunocytochemistry and biotinylation results. These results suggest the existence of cell-specific mechanisms that discriminate between neurotransmitter transporters for surface expression and render unlikely any simple hypothesis that sorting mechanisms in neurons and epithelia are identical.

Animals↗
PubMed1996

Real-time detection of the surface delivery of newly synthesized membrane proteins.

Newly synthesized membrane proteins travel from the Golgi complex to the cell surface in transport vesicles. We have exploited the ion channel properties of the nicotinic acetylcholine receptor (AChR) to observe in real time the constitutive delivery of newly synthesized AChR proteins to the plasma membrane in cultured muscle cells. Whole-cell voltage clamp was employed to monitor the current fluctuations induced by carbamylcholine upon the insertion into the plasma membrane of newly synthesized AChRs, following release from a 20 degrees C temperature block. We find that the transit of vesicles to the cell surface occurs within a few minutes after release of the block. The time course of electrical signals is consistent with many of the fusion events being instantaneous, although some appear to reveal the flickering of a fusion pore. AChR-containing vesicles can fuse individually or as conglomerates. Intracellular application of guanosine 5'-[gamma-thio]triphosphate inhibits the constitutive traffic of AChRs in most cells. Individual exocytotic vesicles carry between 10 and 300 AChR molecules, suggesting that AChRs may be packed extremely densely.

Animals↗
PubMed1996

Polarized expression of GABA transporters in Madin-Darby canine kidney cells and cultured hippocampal neurons.

At least three high affinity Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporters are known to exist in the rat and mouse brain. These transporters share 50-65% amino acid sequence identity with the kidney betaine transporter which also transports GABA but with lower affinity. The betaine transporter (BGT) is expressed on the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. Recent evidence suggests that the signals and mechanisms involved in membrane protein sorting share many functional characteristics in polarized neurons and epithelial cells. It was previously shown that the rat GABA transporter GAT-1 is located in the presynaptic membrane of axons where it plays a role in terminating GABAergic neurotransmission. When expressed in MDCK cells by transfection, GAT-1 was sorted to the apical membrane. In this report, we have localized the other two GABA transporters, GAT-2 and GAT-3, in transfected MDCK cells by GABA uptake, immunofluorescence, and cell surface biotinylation. GAT-3, like GAT-1, localized to the apical membrane of MDCK cells while GAT-2, like BGT, localized to the basolateral membrane. We have also expressed BGT in low density cultures of hippocampal neurons by microinjection and immunolocalized it to the dendrites. The distribution of GAT-3 in these neurons after transfection was axonal as well as somatodendritic. These results indicate that highly homologous subtypes of GABA transporters are sorted differently when expressed in epithelial cells or neurons and suggest that these two cell types share the capacity to distinguish among these isoforms and target them to distinct destinations.

Amino Acid Sequence↗