1. Purification of OT receptors from mammary plasma membranes of lactating rats. The long term goal is to purify receptor and to characterize its structure and function. From preliminary results, a 2- or 3-step procedure should yield pure receptor. These steps include size exclusion and affinity chromatography on ligand and lectin affinity columns. The studies will utilize a newly developed iodinated antagonist of TO, which has an affinity for the receptor more than 200 times greater than TO. The iodinated antagonist- receptor complex remains intact during various manipulations. 2. Determination of complete or partial amino acid N-terminal sequence for validation of cDNA sequence determined from expression vectors (see aim 4), or for construction of oligonucleotide probes for screening of cDNA libraries, if required. 3. Development of monoclonal antibodies to OT receptors for possible use as reagents to immunoaffinity purify receptors, to characterize the subunit structure of the receptor, determine the relatedness of receptors from the mammary gland, uterine myometrium, uterine endometrium and regions of the brain, and to determine the effects of inhibition of TO binding in vivo on the outcome of parturition. 4. Expression of OT receptors in a transient cell culture system: (a) to allow cDNA isolation and sequencing for determination of the primary structure of the receptor, and (b) and as a basis for future studies using site-directed mutagenesis to study structure-function relationships.
