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RL Schubert and D Puett

Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.

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TP Meehan, D Puett and P Narayan

We have previously reported the construction of a constitutively active luteinizing hormone receptor by covalently linking a fused heterodimeric hormone to the extracellular domain of the G protein-coupled receptor. This yoked hormone-receptor complex (YHR) was found to produce high levels of cAMP in the absence of exogenous hormone. Stable lines expressing YHR were generated in HEK 293 cells to obtain lines with different expression levels; however, in a relatively short time of continued passage, it was found that YHR expression was greatly reduced. Herein, we describe the development of clonal lines of HEK 293 cells in which the expression of YHR is under the control of a tetracycline-regulated system. Characterization of clonal lines revealed tight control of YHR expression both by dose and time of incubation with doxycycline. These experiments demonstrated a good correlation between expression levels of the receptor and basal cAMP production. Moreover, the reduction in receptor expression following doxycycline removal revealed that YHR mRNA and protein decayed at similar rates, again suggesting a strong linkage between mRNA and protein levels. The controlled expression of YHR in this cell system will allow for a more detailed analysis of the signaling properties associated with constitutive receptor activation and may prove to be advantageous in developmental studies with transgenic animals.