Carboxyl-terminal sequence of entactin deduced from a cDNA clone.

Entactin is a widely distributed basement membrane sulfated glycoprotein of approximately equal to 150 kDa. The entactin gene is expressed early in mouse embryogenesis. Two cDNA clones complementary to rat entactin mRNA were isolated by antibody screening of an oligo(dT)-primed cDNA library constructed in the lambda gt11 expression vector. One of the clones, lambda 1E, was subcloned into plasmid pBR322 and further characterized. The clone contained sequences complementary to an mRNA species 6 kilobases in length. This mRNA was translated in rabbit reticulocyte lysates to yield a polypeptide of 143 kDa that was precipitated with anti-entactin antiserum. The cDNA insert, 1328 base pairs long, was sequenced and found to contain an open reading frame of 729 base pairs that coded for 243 amino acids at the carboxyl terminus of entactin. Analysis of the peptide revealed no extended alpha-helical or beta-sheet secondary structures. Radiolabeled probes prepared by nicktranslation of p lambda 1E were used to monitor the steady-state levels of entactin mRNA in F9 embryonal carcinoma cells that were induced to differentiate by exposure to retinoic acid and dibutyryl cyclic AMP. The increase in steady-state levels of entactin mRNA lagged behind the increase in mRNA for the B2 chain of laminin, suggesting that laminin and entactin are independently rather than coordinately regulated.

Inhibition of acetylcholine receptor assembly by activity in primary cultures of embryonic rat muscle cells.

Silencing of contractile activity in muscle is known to increase the level of acetylcholine receptor on the cell surface. Both in vivo and in vitro studies indicate that modulation of receptor-specific mRNA levels plays a role in the activity-related regulation, but other mechanisms have not been explored. In this study, we examine the synthesis and post-translational fate of receptor alpha subunit in actively contracting and tetrodotoxin-inhibited rat muscle cultures. Using metabolic labeling and immunoprecipitation with subunit-specific monoclonal antibodies, we find that the increase of alpha subunit synthesis in tetrodotoxin-inactivated cultures is insufficient to account for the increased rate at which new receptors appear on the cell surface. In evaluating stages in the post-translational processing of alpha subunit, we find that in active and inactive cultures, newly synthesized subunit acquires the ability to bind alpha-bungarotoxin with the same kinetics. However, differences were noted at or preceding the stage where alpha subunit becomes assembled with the other subunits to form the 9 S receptor. In inactivated cultures, newly synthesized alpha subunit transits a 5 S precursor pool more rapidly and is assembled more efficiently than in contracting cultures. The possibility that these differences represent a type of post-translational regulation is discussed.

An acetylcholine receptor precursor alpha subunit that binds alpha-bungarotoxin but not d-tubocurare.

We have identified an intracellular form of the alpha subunit of the acetylcholine receptor that binds alpha-bungarotoxin with high affinity. Unlike the mature receptor complex, an alpha 2 beta gamma delta pentamer that migrates as a 9S species in velocity sedimentation analysis, the intracellular species moves as a 5S component. The kinetics of appearance of alpha subunit in the 5S component and the mature receptor complex indicate that the intracellular 5S component is a precursor of the mature receptor. The precursor species differs from 9S receptor in two critical features: (i) the precursor alpha subunit is not associated with beta subunit and (ii) alpha-bungarotoxin binding to the precursor alpha subunit is not inhibited by the cholinergic ligands decamethonium or d-tubocurarine. The properties of the precursor suggest that the acquisition of the ligand binding site by alpha subunit occurs at a distinct stage in the posttranslational development of functional acetylcholine receptor.

Synthesis of laminin and entactin by F9 cells induced with retinoic acid and dibutyryl cyclic AMP.

Mouse embryonal carcinoma F9 cells were exposed to retinoic acid and dibutyryl cyclic AMP. The treated cells synthesized and secreted into the culture medium the basal lamina components, laminin (GP-1 and GP-2) and entactin. The time course of secretion of the basal lamina components was examined by electron microscopic and immunochemical procedures. The induction of the cells resulted in major morphological changes and the deposition of both laminin and entactin at the cell surface and cell junctions. Intracellular deposits of laminin could be localized to the endoplasmic reticulum and membrane-bound intracytoplasmic vacuoles. Concomitant with the appearance of laminin and entactin, there was a loss of fibronectin synthesis and a marked decrease in a 190,000-Da sulfated glycoprotein that appeared to be related to entactin. In the induced cells, laminin and entactin were associated in a complex that could be dissociated with low concentrations of sodium dodecyl sulfate. The induction of laminin and entactin seem to be independent. The enhanced synthesis of laminin appeared to be under transcriptional regulation since it was found that induced F9 cells contained translatable mRNA for GP-2 when tested in a rabbit reticulocyte lysate system. The uninduced cells did not contain detectable quantities of translatable GP-2 mRNA.