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 rece ptors typified by the growth hormone receptor, A common structural element found in cytokine receptors is a module of two fibronectin-like domains, ea ch characterized by seven beta-strands denoted A-G and A'-G', respectively. The alpha-chain (GMR alpha) confers low affinity GM-CSF binding (K-d = 1-5 nM), whereas the beta-chain (beta(c)) does not bind GM-CSF by itself but c onfers high affinity binding when associated with alpha (K-d = 40-100 pM), In the present study, we define the molecular determinants required for lig and recognition and for stabilization of the complex through a convergence of several approaches, including the construction of chimeric receptors, th e molecular dynamics of our three-dimensional model of the GM.GMR complex, and site-directed mutagenesis. The functional importance of individual resi dues was then investigated through ligand binding studies at equilibrium an d through determination of the kinetic constants of the GM.GMR complex, Cri tical to this tripartite complex is the establishment of four noncovalent b onds, three that determine the nature of the ligand recognition process inv olving residues Arg(280) and Tyr(226) Of the alpha-chain and residue Tyr(36 5) Of the beta-chain, since mutations of either one of these residues resul ted in a significant decrease in the association rate. Finally, residue Tyr 365 Of beta(c) serves a dual function in that it cooperates with another re sidue of beta(c) Tyr(421) to stabilize the complex since mutation of Tyr(36 5) and Tyr421 result in a drastic increase in the dissociation rate (Koff), Interestingly, these four residues are located at the B'-C' and F'-G' loop s of GMR alpha and of beta(c), thus establishing a functional symmetry with in an apparently asymmetrical heterodimeric structure.