COMBINATION OF THE CHORIONIC-GONADOTROPIN FREE BETA-SUBUNIT WITH ALPHA

The rate-limiting event for combination of hCG α- and β-subunits in JAR choriocarcinoma cells is the rate of disulfide bond formation in the β -subunit. This is accompanied by a conformational change that produces a combination-com-petent form of the β -subunit. The combination reaction, how-ever, is incomplete, and 50% of the synthesized β molecules remain uncombined (free). In addition, 70% of biosynthetically labeled free β is degraded in the cell. Possible explanations for incomplete dimer formation include 1) biochemical differences between the free and combined β -subunits that limit combina-tion of free β, and 2) an inefficient combination reaction due to low intersubunit affinities or limiting concentrations of combination-competent subunits within the cell. To examine whether the biochemical differences between free and combined β -subunits that we have previously observed affect the combination competence of free β, free and dimer β -subunits were purified from the culture medium and lysates of JAR cells and examined for their ability to combine with α purified from pregnancy urine in an in vitro combination assay. Secreted free and dimer β obtained from culture medium combined to the same extent with urinary α. Although the combination efficiencies were lower for the intracellular forms, the free and dimer β - subunits purified from cell lysates also combined to the same extent with urinary α. Thus, biochemical differences that exist between the β forms do not prevent combination of free β with a in an in vitro combination assay. To examine the second possibility, we speculated that if high concentrations of hCG subunits remained in the rough endoplasmic reticulum (ER) for extended periods of time, the extent of dimer formation would increase in the cell. To increase the residence time of hCG subunits in the ER, JAR cells were treated with carbonyl cyanide trifluoromethoxyphenylhydrazone, an agent that inhibits the translocation of hCG subunits from the ER to the Golgi. Treatment of cells with trifluoromethoxyphen- ylhydrazone in long and short term pulse-chase labeling studies did not result in an increase in the extent of dimer formation. Thus, the subunit combination reaction in JAR cells may be incomplete due to subtle conformational differences in the free β -subunit; however, these differences do not inhibit the combination of the free β -subunit in vitro. © 1990 by The Endocrine Society.