Mathematical Model of Human Growth Hormone (hGH)-Stimulated Cell Proliferation Explains the Efficacy of hGH Variants as Receptor Agonists or Antagonists

Abstract

Human growth hormone (hGH) is a therapeutically important endocrine factor that signals various cell types. Structurally and functionally, the interactions of hGH with its receptor have been resolved in fine detail, such that hGH and hGH receptor variants can be practically engineered by either random or rational approaches to achieve significant changes in the free energies of binding. A somewhat unique feature of hGH action is its homodimerization of two hGH receptors, which is required for intracellular signaling and stimulation of cell proliferation, yet the potencies of hGH mutants in cell-based assays rarely correlate with their overall receptor-binding avidities. Here, a mathematical model of hGH-stimulated cell signaling is posed, accounting not only for binding interactions at the cell surface but induction of receptor endocytosis and downregulation as well. Receptor internalization affects ligand potency by imposing a limit on the lifetime of an active receptor complex, irrespective of ligand-receptor binding properties. The model thus explains, in quantitative terms, the numerous published observations regarding hGH receptor agonism and antagonism and challenges the interpretations of previous studies that have not considered receptor trafficking as a central regulatory mechanism in hGH signaling.

A Bipolar Clamp Mechanism for Activation of Jak-Family Protein Tyrosine Kinases

“…Using our Simplified Cellular Model (Methods), we quantified activated (receptor-bound and Y2-phosphorylated) Jak2 stimulated by varying doses of GH at steady state, relative to the number of cell-surface GH receptors in the absence of GH (Figure 3); as explained previously [24], maximal GH receptor activation is accompanied by significant downregulation from the surface, so a relative value of ∼0.05 by this measure is the maximum. The Simplified Cellular Model does not allow for membrane localization of SH2-Bβ through its PH domain.…”

“…Where applicable, we build upon a previous model of GH/GH receptor interactions and trafficking [24] and use the same parameter values for wild-type human GH. Briefly, the GH ligand concentration [ L ] is fixed and is an input variable to the model, and unbound GH receptors ( R ) are present at a level of 2×10 3 molecules/cell initially.…”

Cellular level models as tools for cytokine design

“…Due to the potentially nonlinear dependence of the number of states and parameters on the topology of the model in such complex systems, simplifying assumptions often have to be made as to the mechanisms of activation or the trafficking kinetics of each receptor dimer. As another example, systems with a 1:2 ligand:receptor stoichiometry that have one high-affinity site and one lower-affinity site may show a peaked dose response curve because 1:1 inactive complexes are favored at higher ligand concentrations [53–55]. In a model that explicitly considered the sequential binding of the 1:2 human growth hormone (hGH) – hGH receptor complex [53], modest increased activity in the hGH system could be realized by increasing ligand–receptor affinity via the on rate through mutation (no gains could be made by decreasing the off rate).…”

“…As another example, systems with a 1:2 ligand:receptor stoichiometry that have one high-affinity site and one lower-affinity site may show a peaked dose response curve because 1:1 inactive complexes are favored at higher ligand concentrations [53–55]. In a model that explicitly considered the sequential binding of the 1:2 human growth hormone (hGH) – hGH receptor complex [53], modest increased activity in the hGH system could be realized by increasing ligand–receptor affinity via the on rate through mutation (no gains could be made by decreasing the off rate). These findings are in agreement with the principles demonstrated here when one recognizes that the model assumed constant ligand concentration and used an off rate that was similar to the internalization rate, such that the system was far more activation-limited than ligand-limited.…”

Discovery of Improved EGF Agonists Using a Novel In Vitro Screening Platform

“…7,8 In many cases, a protein's binding affinity toward, for example, a receptor does not correlate with biological efficacy, and maximizing affinity can even be counterproductive. [9][10][11] This is due to myriad additional factors that influence cell signaling, including binding kinetics, receptor internalization, and intracellular protein trafficking. 12,13 Thus, there is a great need for an efficient, general method to screen libraries of mutant proteins for overall biological function.…”