Journal of Biological Chemistry · 1990 · 235 citations · 29 references
Life SciencesBiochemistryMolecular BiologyCell SignalingMolecular Cell BiologyProtein-protein InteractionsCell-surface ReceptorsHormone Binding ProteinsLigand BindingMembrane ReceptorsReceptor BindingReceptor PhysiologyG Protein-coupled Hormone ReceptorsG-protein-coupled ReceptorsHomologyHormone Binding AffinityHormone Receptor Binding ProteinsMembrane Hormone ReceptorsMolecular HomologyReceptors
The lutropin-choriogonadotropin (LH/CG) receptor is a cell surface receptor comprised of two domains of roughly equivalent size. The amino-terminal half of the receptor is relatively hydrophilic and is located extracellularly, whereas the carboxyl-terminal half of the receptor shares amino acid homology with other receptors that couple to G proteins and is similarly thought to span the plasma membrane seven times, ending with a relatively short carboxyl-terminal tail. In order to test the role of the extracellular domain in binding hormone, we constructed a mutated rat luteal LH/CG receptor cDNA (termed pCLHR-D2), which encodes for only the extracellular domain, and used it to transiently transfect human kidney 293 cells. Here we report that the expressed extracellular domain of the LH/CG receptor is capable of binding human CG with a high affinity, comparable with that of the full-length receptor. Thus, not only is the extracellular domain of the glycoprotein hormone receptors involved in binding hormone, but it alone is capable of conferring high affinity binding. Unexpectedly, it was also found that this truncated receptor is not secreted into the culture media but remains trapped within the cells.
29
Site-directed mutagenesis by overlap extension using the polymerase chain reaction
Steffan N. Ho, Henry D. Hunt, Robert M. Horton et al. · Gene · 1989 · 7.5K citations