A membrane protein associated with the prolactin receptor studies with a photoactivatable human growth hormone derivative

Prolactin receptor from rat liver (PRL-R, 42 kDa) was cross-linked to a radiolabeled azidophenacyl derivative of human growth hormone ([ 125I]AP-hGH) to yield a 63 kDa adduct. In addition, a protein of Mr 50–52 K was detected as a 73 kDa complex. Microsomes incubated with either (a) increasing amounts of [ 125I]AP-hGH, or (b) a fixed amount of photoprobe and increasing concentrations of unlabeled hGH, showed that the 73/63 kDa band intensity ratio remains constant (0.71–0.77). Once transferred onto nitrocellulose membranes, only the 42 kDa protein is able to bind [ 125I]AP-hGH or [ 125I]hGH. Two anti-PRL-R monoclonal antibodies fail to cross-react with proteins of Mr 50–52 K. In membranes solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a significantly lower amount of the 73 kDa complex is detected. Thus, the 50–52 kDa protein appears to be structurally unrelated to, but is presumably associated with the PRL-R. The 73 kDa complex is also detected under low membrane fluidity conditions (1 °C), indicating that PRL-R associates to this 50–52 kDa protein prior to hormone binding. Perfusion of rat liver with [ 125I]AP-hGH shows that this associated protein accompanies the receptor along its intracellular pathway.