The G protein-coupled receptor superfamily represents one of the largest gene families in vertebrates. Among this group of proteins, the thyrotropin and gonadotropin receptors represent a subfamily with important functions in thyroid and gonads, respectively. These glycoprotein hormone receptors are unique in having leucine-rich repeat-containing ectodomains responsible for ligand binding. Advances in genome projects have allowed the discovery of many novel genes. Based on sequence similarity searches, we have identified genes encoding five human, one nematode, and four fly orphan receptors sharing the conserved domain arrangement found in mammalian glycoprotein hormone receptors and named them as LGRS (Leucine-rich repeat containing, G protein-coupled Receptors). The mammalian orphan LGRS can be divided into two subgroups and our studies focus on one prototypic member, LGR4. We propose to characterize the ovarian expression pattern and putative ligands of this novel receptor to understand its physiological functions. Because glycoprotein hormone receptors are among the few GPCRs in which the ligand-binding region resides in the ectodomain, the leucine-rich repeats in the homologous LGR ectodomains are likely to be the ligand-binding regions of these orphan LGRs. We will generate the soluble ectodomain of LGR4 for the characterization of its putative ligand(s). Preliminary characterization of an LGR4 ectodomain-splicing variant allowed the generation of large amounts of this soluble ligand-binding region. We have already purified mg quantities of the LGR4 splicing variant encoding the putative ligand-binding region and generated antibodies against this protein for immunocytochemical localization of LGR4 in different ovarian cells. Northern blot analysis also demonstrated the expression and gonadotropin regulation of LGR4 mRNAs in the ovary. The soluble ligand-binding region for LGR4 will be used as a "reverse ligand" for the identification and affinity purification of putative endogenous agonists for this orphan receptor and our preliminary data indicate that co-treatment with this protein attenuated the gonadotropin stimulation of ovarian weight gain in vivo. We propose to set up a granulosa cell-based bioassay, coupled with affinity purification, to characterize the ligand(s) for LGR4 in follicular fluid and ovarian extracts in this pilot application. Eventual purification of the ligand(s) for LGR4 could provide understanding of its physiological roles in ovary and other tissues.