Human lactogenic hormones, including growth hormone (hGH), placental lactogen (hPL) and prolactin (hPRL), all bind the human lactogen receptor. Current data suggests that lactogen and receptor bind with a 1:2 ratio and the two binding reactions have affinities that differ by at least an order of magnitude. Current information suggests lactogens may be similar to hGH and the somatotropin receptor where binding is an ordered process. The ordered hGH mechanism was demonstrated by mutagenic studies, where mutation of Site 1 eliminated binding at Site 2, but mutation of Site 2 had no effect of Site 1 binding. These results documented a uni-directional interaction between the two somatotrophic sites of hGH. Similar studies have been difficult to perform with lactogens because of the low affinity of Site 2. In fact, until examined by surface plasma resonance techniques, binding at Site 2 has been difficult to consistently document. No detailed structural mechanism has been described that will account for the interaction between Sites 1 and 2 in any lactogen. Using biological assays for lactogenic activity, we have recently described a series of contiguous hydrophobic residues in hGH that may be the motif by which Site 1 interacts with Site 2. Mutation of these residues, which span from Site toward Site 2, severely reduced the activity at Site 2. The main goal of this application is to determine if this motif is responsible for the interaction between Sites 1 and 2 in hGH, hPL and hPRL. Specific Objective 1 will characterize and compare the nature of lactogen/receptor binding; determining if the two binding sites interact and the nature and degree of interaction. Characterization of the binding mechanism of hGH, hPL and hPRL must be completed before studies probing the structural motif can be interpreted. Specific Objective 2 will determine the physical nature of the motif that apparently conducts the communication between Sites 1 and 2 of hGH. These studies will determine the size of the motif and characterize the role of each member residue in the communication between the two receptor binding sites. Specific Objective 3 will determine the same or a similar motif is conserved and operation in hPL and hPRL.
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