Only Multimeric Hensin Located in the Extracellular Matrix Can Induce Apical Endocytosis and Reverse the Polarity of Intercalated Cells

Abstract

When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H ؉ -ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35 Slabeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.The plasma membrane of epithelial cells is polarized into two domains; apical and basolateral, each of which has characteristic protein and lipid composition (1-3). Although targeting sequences were found in the structure of several polarized proteins, recent studies have shown that some proteins are polarized in a cell type-specific manner; for instance the Na,KATPase is targeted to the apical membrane of retinal pigment epithelium but to the basolateral membrane of most other epithelia (4). Several other proteins exhibit this flexibility in targeting; the most dramatic example is the targeting of the proton-translocating ATPase and the Cl:HCO 3 exchanger of the intercalated cell of the renal tubule (reviewed in Ref. 5). These cells exist in a spectrum of forms. One extreme, the ␣ type, has an apical H ϩ -ATPase and a basolateral anion exchanger that is an alternately spliced form of the erythroid band 3 (kAE1) 1 and hence is capable of trans-epithelial secretion of H ϩ . In contrast, the ␤ form secretes HCO 3 by a basolateral H ϩ -ATPase and an apical kAE1 (6). (However, one study did not find kAE1 by immunoblot analysis but confirmed the presence of its mRNA (7).) In an...

Acid incubation reverses the polarity of intercalated cell transporters, an effect mediated by hensin

“…Ab's against SRCR domains 6 and 7 prevented the development of the high-density phenotype and inhibited apical endocytosis 50-70% (18,19). Similarly, these Ab's significantly reduced the adaptive inhibition of HCO 3 -secretory flux by an equivalent fraction, but completely prevented the appearance of basolateral Cl -/HCO 3 -exchange and the disappearance of apical anion exchange.…”

“…Previous studies suggested that this reduction in cap length is a consequence of endocytosis of these apical markers. Studies in vivo (4,5) and in the intercalated cell line in vitro (18,24) had demonstrated that induction of apical endocytosis is one of the first phenomena to occur during conversion of β-to α-intercalated cell phenotypes. These findings suggest that endocytic removal of apical Cl -/HCO 3 -exchangers results in reduction of apical membrane area of β-intercalated cells.…”

Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

“…17 In addition, hensin is present in a pattern compatible with ECM deposition in the mature columnar epithelia of the intestinal villus and prostate luminal cells but not in the less differentiated epithelia of the crypt of the small intestine and basal cells of the prostate. 5 Preliminary studies with negative staining electron microscopy show that ECM hensin forms 50-to 100-nm-long fibers composed of several fibrils (S.V.…”

“…and Q.A.-A., unpublished observations). Hensin seems to undergo a complex polymerization path that requires the presence of at least two other proteins, galectin3 and a cis-trans peptidyl prolyl isomerase [17][18][19] ; however, when 35 S-labeled monomeric hensin was added to high-density cells, it became polymerized, suggesting that a cellsurface event (similar to what occurs during fibronectin fibril formation 20 and not a biosynthetic intracellular event (e.g., that seen in collagen assembly 21 ) causes the polymerization of hensin. Here we purified receptor proteins that bind hensin and found that they were integrins.…”

2007 Homer W. Smith Award

“…Purified hensin from the ECM has a very high molecular weight (in excess of 10 mil- lion), and negative-staining electron microscopy shows that it is composed of fibers each of which is composed of many fibrils. Extensive biochemical studies by Chinami Hikita 18 showed that hensin is secreted as a monomer in the ␤ intercalated cell but undergoes a complex polymerization in the extracellular space, which leads to the formation of its insoluble ECM form. Remarkably, monomeric hensin is not functional; only hensin in insoluble ECM is capable of inducing the ␤ to ␣ intercalated plasticity.…”