Kinetic analysis of receptor-mediated endocytosis of G-CSF derivative, nartograstim, in rat bone marrow cells.
To elucidate the mechanism of the receptor-mediated clearance of granulocyte colony-stimulating factor (G-CSF), we performed kinetic analyses of the receptor-mediated endocytosis (RME) processes using a human G-CSF derivative, nartograstim (NTG), and isolated rat bone marrow cells. The first-order rate constants involved in RME processes were obtained by computerized model fitting of the time courses of the ligand-receptor complex on both the cell surface and in the cell interior and the degradation products in the medium in the pulse-chase experiment. They were also calculated based on a kinetic model involving the ligand concentration dependence of the initial binding rate, the steady-state degradation rate, and the steady-state amounts of ligand on both the cell surface and in the interior. The rate constants for the RME processes after receptor binding determined in the different experiments were similar, that is, the half-times for the dissociation, internalization, and degradation of the ligand-receptor complex were 770, 10-30, and 20 min, respectively. However, the association constant obtained by measuring the initial binding was fivefold greater than that calculated under steady-state conditions. These kinetic analyses support the hypothesis that the internalization of the receptor may be accelerated by ligand binding, causing downregulation of the receptor on the cell surface. These overall kinetic analyses based on steady-state and non-steady-state data of the RME processes clarify the dynamics of the interaction between NTG and its receptor.
Kuwabara
T
T
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Company, Shizuoka, Japan.
Kobayashi
S
S
Sugiyama
Y
Y
D000818:Animals; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D015536:Down-Regulation; D004705:Endocytosis; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D008297:Male; D008954:Models, Biological; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface / Q000502:physiology*; D013696:Temperature
D011956:Receptors, Cell Surface; D016179:Granulocyte Colony-Stimulating Factor; C072115:nartograstim