Human growth hormone-stimulated growth of human cultured lymphocytes (IM-9) and its inhibition by phorbol diesters through down-regulation of the hormone receptors. Possible involvement of phosphorylation of a 55,000 molecular weight protein associated with the receptor in the down-regulation.
We report in this paper that human growth hormone (hGH) stimulates the growth of human cultured IM-9 lymphocytes in a low concentration (3%) of serum. The hormone-stimulated growth was inhibited with the phorbol diesters phorbol 12-myristate 13-acetate and phorbol 12,13-dibutylate (PDBu). The binding experiments of 125I-hGH to the phorbol diester-treated cells and to their detergent-solubilized receptors revealed that the phorbol diesters caused internalization of the hGH receptors from the cell surfaces but did not significantly affect their affinity (Ka = 8.5 x 10(9) M-1). About half of the receptors (1.4 x 10(3)/cell) were internalized in 30 min at 37 degrees C, and the half-effective doses of phorbol 12-myristate 13-acetate and PDBu were 5 and 35 nM, respectively. When culture was continued after washing with the culture medium, the phorbol diester-treated cells recovered their hGH-responsive growth, and the number of the surface hGH receptors was restored. The down-regulation of the hormone receptor was also induced with another phorbol diester, phorbol 12,13-didecanoate, but not with the phorbol or phorbol monoesters phorbol 12-myristate and phorbol 13-acetate. The synthetic activators of protein kinase C 1-oleoyl-2-acetyl-glycerol and N-(6-phenyl-hexyl)-5-chloro-1-naphthalenesulfonamide had an effect similar to that of the phorbol diesters. Staurosporine and sphingosine, inhibitors of protein kinase C, inhibited the phorbol diester-caused down-regulation with a half-inhibitory dose (IC50) of 8 nM and 130 microM, respectively. This suggests that protein kinase C was involved in the reaction. When 32Pi-loaded IM-9 cells were stimulated with PDBu at 37 degrees C, the phosphorylation of Mr 55,000, 88,000, and 114,000 proteins increased rapidly. The PDBu-stimulated phosphorylation of 55,000 protein was also inhibited by staurosporine at 10 nM, which was a comparable concentration to inhibit the phorbol diester-induced down-regulation of hGH receptors. Furthermore, among these proteins, the 55,000 protein was specifically coisolated with the hGH receptors by three different experiments: 1) immunoprecipitation by anti-hGH antibody; 2) immunoisolation using protein A-cellulose columns; and 3) affinity purification by hGH-fixed agarose gel. These results suggest that phorbol diesters reduce the hGH-stimulated growth of cultured IM-9 lymphocytes by the down-regulation of hGH receptors and that the receptor-associated 55,000 protein may be involved in this regulation through phosphorylation by protein kinase C.
Suzuki
K
K
Division of Biochemistry and Immunochemistry, National Institute of Hygienic Sciences, Tokyo, Japan.
Suzuki
S
S
Saito
Y
Y
Ikebuchi
H
H
Terao
T
T
D000470:Alkaloids / Q000494:pharmacology; D001769:Blood; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology*; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D013110:Sphingosine / Q000494:pharmacology; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D010703:Phorbol Esters; D010750:Phosphoproteins; D011986:Receptors, Somatotropin; C008876:phorbol-12,13-didecanoate; D015240:Phorbol 12,13-Dibutyrate; D013006:Growth Hormone; D011493:Protein Kinase C; D019311:Staurosporine; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate