Site-directed mutagenesis of hGH at the 54-74 loop selectively modifies its lactogenic receptor-mediated biological activity

Four analogues of human growth hormone (hGH) mutated by site-directed mutagenesis at the 54-74 loop-MethGH(P59A), Met-hGH(P6lA), Met-hGH(P59A,P61A) and Met-hGH(Des 62-67) were analyzed for: (1) their biological activity mediated through lactogenic receptors using rat lymphoma Nb₂-llC cell proliferation and mouse mammary gland HC-11 cell β-casein synthesis bioassays and (2) their ability to interact with recombinant hGH binding protein (hGHBP). The analogues Met-hGH(P59A), Met-hGH(P61A) and Met-hGH(P59A,P61A) partially lost their activity relative to native hGH in the HC-11, but not in the Nb₂-llC cell bioassay. These analogues were nevertheless capable of forming a 1:2 complex with a recombinant hGH binding protein (hGHBP), despite the fact that the affinity of Met-hGH(P61A) and Met-hGH(P59A,P61A) analogues had decreased 8- and 14-fold, respectively. Met-hGH(Des 62-67) failed to form 1:1 or 1:2 complexes with hGHBP and did not compete with [¹²⁵I]hGH for binding to hGHBP. It lost all biological activity in HC-11 cells, but retained 0.4% of its activity, in the Nb₂-11C cell proliferation bioassay. These results confirm the involvement of Pro-61 in the hGH binding and activity mediated through somatogenic receptors, while the activity mediated through two different types of lactogenic receptors was selectively modified. These findings emphasize the fact that lactogen receptors in different species or organs are not identical.