The influences of hyperprolactinaemia and obesity on cardiovascular risk markers: effects of cabergoline therapy

© 2006 Blackwell Publishing Ltd, Clinical Endocrinology , 65 , 826–831 detected by an Enhanced Chemiluminescence System (GE Healthcare, NJ, USA). Transfection with the wild-type CASR showed monomeric forms of 140 and 160 kDa and other higher molecular weight species corresponding to dimeric and oligomeric forms. The most abundant were the monomeric partial glycosylated species of 140 kDa, and the dimeric and oligomeric forms (Fig. 1c). Compared to the wild-type, expression levels of the mutant receptor containing the mutation in the start site were reduced dramatically (Fig. 1c), indicating an abnormality in processing of the receptor. Immunocytochemistry was used to identify the cellular localization of the truncated receptor. HEK-293 cells transiently transfected with the wild-type and mutant receptor were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) plus 0·12 sucrose. For permeabilization, cells were further treated with 0·2% Triton X-100 in PBS and 0·1% of Triton X-100 was added to all washes and antibody incubations. Strong staining was observed at the cell surface of nonpermeabilized and permeabilized HEK293 cells expressing the wild-type receptor (Fig. 1d). By contrast, the mutant receptor was not expressed on the cell surface and staining was only identified inside the permeabilized cells, suggesting that the truncated receptor was trapped within the endoplasmatic reticulum (Fig. 1d). The dramatic reduction in expression of the mutated receptor and the fact that the antibody used identified the mutant receptor are likely to be due to leaking of translation of the mutant ATG. Another less likely event is that the mutation results in activation of the cryptic translational initiation site in exon 3 in frame, preserving the epitope from which the antibody was raised (amino acids 315–335). This would result in a truncated protein without the original amino terminal, 10 kDa shorter (p.M1_A73del). We were not able to identify differences in size compared to the wild type as the protein is poorly expressed; however, based on the scanning model, this site of translational initiation is too far away from the 5 ′ cap to be used, and is in a suboptimal context. 6