Deletions of Portions of the Extracellular Loops of the Lutropin/Choriogonadotropin Receptor Decrease the Binding Affinity for Ovine Luteinizing Hormone, but Not Human Choriogonadotropin, by Preventing the Formation of Mature Cell Surface Receptor

The rat lutropin/choriogonadotropin receptor (rLHR) is a G protein-coupled receptor which binds either human choriogonadotropin (hCG) or lutropin (luteinizing hormone, LH) and, therefore, plays a central role in reproductive physiology. In addition to the seven transmembrane helices, three extracellular loops, three intracellular loops, and a cytoplasmic tail characteristic of all G protein-coupled receptors, the rLHR also contains a relatively large N-terminal extracellular domain. Since high affinity hormone binding occurs to this N-terminal extracellular domain and since G proteins are activated by intracellular regions of the receptor, it has been hypothesized that upon hormone binding a portion of the hormone or the receptor's extracellular domain might interact with the receptor's extracellular loops and/or transmembrane helices, thus evoking an intracellular conformational change. To explore this possibility, we prepared and characterized several mutants of the rLHR in which portions of the extracellular loops were deleted. Ultimately, it was not possible to examine the signal transduction properties of the mutants because all but one mutant were retained intracellularly. Although the intracellularly retained mutants must be somewhat misfolded, all were found to bind hCG with high affinity if the cells were first solubilized in detergent. However, the binding of oLH to the detergent solubilized mutants was altered. Thus, whereas the wild-type rLHR bound oLH with two apparent affinities, the solubilized deletion mutants bound oLH with only one apparent affinity. Although these data could be interpreted to suggest that an ovine LH (oLH) binding site on the extracellular loops of the rLHR was deleted, data shown argue against this hypothesis. Rather, the results presented suggest that the two apparent affinities of the wild-type rLHR for oLH represent the binding affinities of two populations of rLHR where the mature, cell surface form binds oLH with a higher affinity than the immature, intracellular form. Furthermore, we show that mutations of the rLHR which cause intracellular retention of the receptor result in a decrease from two to one apparent binding sites for oLH due to the absence of the high affinity oLH binding component contributed by the mature cell surface receptor. Therefore, whereas hCG cannot discriminate between the mature cell surface wild-type receptor and an intracellularly retained rLHR mutant, oLH can make this discrimination, thus suggesting a conformational difference between the two forms of the receptor.