Molecular Determinants of the Granulocyte-Macrophage Colony-stimulating Factor Receptor Complex Assembly

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is composed of two chains that belong to the superfamily of cytokine receptors typified by the growth hormone receptor. A common structural element found in cytokine receptors is a module of two fibronectin-like domains, each characterized by seven β-strands denoted A–G and A′–G′, respectively. The α-chain (GMRα) confers low affinity GM-CSF binding ( K d = 1–5 n m ), whereas the β-chain (β c ) does not bind GM-CSF by itself but confers high affinity binding when associated with α ( K d = 40–100 p m ). In the present study, we define the molecular determinants required for ligand recognition and for stabilization of the complex through a convergence of several approaches, including the construction of chimeric receptors, the molecular dynamics of our three-dimensional model of the GM·GMR complex, and site-directed mutagenesis. The functional importance of individual residues was then investigated through ligand binding studies at equilibrium and through determination of the kinetic constants of the GM·GMR complex. Critical to this tripartite complex is the establishment of four noncovalent bonds, three that determine the nature of the ligand recognition process involving residues Arg 280 and Tyr 226 of the α-chain and residue Tyr 365 of the β-chain, since mutations of either one of these residues resulted in a significant decrease in the association rate. Finally, residue Tyr 365 of β c serves a dual function in that it cooperates with another residue of β c , Tyr 421 to stabilize the complex since mutation of Tyr 365 and Tyr 421 result in a drastic increase in the dissociation rate (Koff). Interestingly, these four residues are located at the B′-C′ and F′-G′ loops of GMRα and of β c , thus establishing a functional symmetry within an apparently asymmetrical heterodimeric structure.