EVIDENCE FOR A 2ND RECEPTOR-BINDING SITE ON HUMAN PROLACTIN

The existence of a second receptor binding site on human prolactin (hP RL) was investigated by site-directed mutagenesis. First, 12 residues of helices 1 and 3 were mutated to alanine. Since none of the resultin g mutants exhibit reduced bioactivity in the Nb2 cell proliferation bi oassay, the mutated residues do not appear to be functionally necessar y. Next, small residues surrounding the helix 1-helix 3 interface were replaced with Arg and/or Trp, the aim being to sterically hinder the second binding site. Several of these mutants exhibit only weak agonis tic properties, supporting our hypothesis that the channel between hel ices 1 and 3 is involved in a second receptor binding site. We then an alyzed the antagonistic and self-antagonistic properties of native hPR L and of several hPRLs analogs altered at binding site 1 or 2. Even at high concentrations (similar to 10 mu M), no self-inhibition was obse rved with native hPRL; site 2 hPRL mutants self-antagonized while site 1 mutants did not. From these data, we propose a model of hPRL-PRL re ceptor interaction which slightly differs from that proposed earlier f or the homologous human growth hormone (hGH) (Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., and Goeddel, D. V., and Well, J. A. (199 2) Science 256, 1677-1680). Like hGH, hPRL would bind sequentially to two receptor molecules, first through site 1, then through site 2, but we would expect the two sites of hPRL to display, unlike the two bind ing sites of hGH, about the same binding affinity, thus preventing sel f-antagonism at high concentrations.