The common α-subunit of the glycoprotein hormones (GP-α) is formed in the pituitary and the placenta, but also by several tumors and under certain pathophysiologic conditions. It was suggested that GPα has growth-regulatory properties and influences differentiation, e.g. in the prostate, since it belongs to the family of cystine-knot proteins, like TGFβ or NGF. We have investigated the biosynthesis of GPα in JEG-3 cells (α and hCG-β formed) compared to HeLa cells (only α-subunit formed) using pulse-chase experiments. Radiolabeled GP-α was precipitated from cell lysates with monoclonal antibodies against α4 and α6 epitopes. In SDS-PAGE (unreduced samples), several GPα isoforms were detected with app. Mr of 19 kD, 22 kD, 35 kD and 40 kD (strong bands underlined) when precipitated with the anti α4-antibody, whereas the 35 and 40 kD forms were not recognized by the anti α6 antibody. In contrast, in JEG-3 cells the 19 and 22 kD forms were the only isoforms detected by both antibodies. Under reduced conditions, the 35 kD and 40 kD bands collapsed into the 19 and 22 kD bands in SDS-PAGE. No apparent differences in the carbohydrate parts between the 22 and 35 kD bands could be detected. During biosynthesis the 35 kD band accumulated continuously (increase of the ratio of 35 /22 kD bands). The different GPα forms are further characterized by MALDI-TOF. Our experiments show that the GPα most probably may form dimers by association in the region of the loop 2 of GPα where the α6 epitope is located. In consequence this epitope will be masked. Depending on the antibody used, this may result in an underestimation of the amount of GPα formed, which is critical when GPα is used as a diagnostic marker. Presently it is unclear why dimer formation did not take place in the JEG cells. Further investigations will be necessary to clarify to what extent GPα monomers and dimers exert different effects on growth and differentiation.
