apical surface forms the bile canaliculi and is specialized for We have identified B10, a plasma membrane protein bile secretion. A variety of apical and basolateral domainpreviously defined by a monoclonal antibody, as an alkaspecific proteins have been identified, which carry out the line phosphodiesterase I (APDE) expressed in the unique functions of each plasma membrane domain. 1 B10, an plasma membrane of rat hepatocytes and enterocytes, apical hepatocyte plasma membrane protein, was originally with a restricted apical distribution. B10 complemenidentified by monoclonal antibodies generated to rat hepatotary DNA (cDNA) was cloned from a rat intestinal library cytes. 2 Its exclusive apical location was shown to be altered screened with a polyclonal antibody directed to the hein the fetus and the young rat, 3 during intrahepatic and expatic protein. Two distinct B10 clones with an open readtrahepatic cholestasis, 4 and after microtubule disruption. 4,5 ing frame of 2,625 bp were obtained that differed only Changes in B10 expression were observed in chemically inby 12 bases in the coding region. One B10 clone had a duced liver carcinomas, 6,7 and in hepatoma cell lines. 8 Howsingle base difference with gp130 RB13-6 cDNA, which was ever, it was not possible to evaluate the consequences of such recently cloned in rat fetal brain. B10/gp130 RB13-6 had 50% disorders because B10 differed from proteins already identiidentity at the amino acid level with the plasma cell antified at the bile canalicular surface, and its function remained gen PC-1, an APDE cloned in the mouse and in human.
unknown.
Anti-B10 antibodies immunoprecipitated 34% of the
In this report, we describe the identity of B10 with APDE activity in liver plasma membranes and over 95% gp130 , a recently cloned fetal brain protein that is also of the APDE activity in intestinal cells. Most of the reexpressed by brain tumor cells but is absent from normal maining activity in hepatocytes (44%) could be immunoadult brain cells. 9 We show that B10 is a member of the precipitated by antibodies directed to PC-1. APDE activfamily of alkaline phosphodiesterase I (APDE) enzymes, the ity immunoprecipitated with anti-B10 antibodies was first member of which was the plasma cell antigen PC-1, a found in the apical rat liver plasma membrane fractions membrane glycoprotein cloned in the mouse 10 and in huon a sucrose gradient whereas most of the remaining man. 11 PC-1 was originally characterized as an antigen that APDE activity was associated with the basolateral fracis selectively expressed on the surface of B lymphocytes when tions, which contained PC-1. By immunofluorescence, they reach their final stage of differentiation into antibody-B10 was localized to the apical surfaces of hepatocytes secreting cells. 12 We show that B10 is the major APDE of and enterocytes whereas PC-1 was present on the basointestinal epithelial cells, whereas in hepatocytes, there are lateral surfaces of hepatocytes. B10/gp130 RB13-6 and rat at least two main APDE enzymes, B10 and PC-1. These two PC-1 are a unique example of distinct molecules having
APDEs are expressed at opposite surfaces of the cells, B10 on similar enzymatic activity but different apical/basolatthe apical membrane and PC-1 on the basolateral, suggesting eral location, and possibly different functions. (HEPATOLthat they may have domain specific functions. OGY 1997;25:995-1002.)
Methods
The hepatocyte plasma membrane is made up of three sur-Animals and Immunological Reagents. Male adult Sprague-Dawfaces. The basal surface faces the blood and has uptake and ley rats weighing 180 to 220 g were obtained from Charles River secretory functions, the lateral surface faces other cells and (Saint-Aubin-le `s Elbeuf, France). All animals received human care is involved in cell-cell adhesion and communication, and the in compliance with the institution's guidelines. The monoclonal anti- body to B10 has been described, 2 as has the polyclonal. 13 The monoclonal antibody 4H4 was directed toward the conserved Ca 2/ binding Abbreviations: APDE, alkaline phosphodiesterase I; IgG, immunoglobulin G; cDNA, region of PC-1. 14 The polyclonal antibody to PC-1 was raised against complementary DNA; mRNA, messenger RNA; SDS, sodium dodecyl sulfate; endo F, endomouse PC-1 and was shown to cross-react with the rat. 15 The polyb-N-acetylglucosaminidase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphateclonal antibody to CE9, a plasma membrane protein located on the buffered saline. basolateral surface of rat hepatocytes 16 was a gift of A. Hubbard (The From