Haman A, Cadieux C, Wilkes B, Hercus T, Lopez A, Clark S, Hoang T
The Clinical Research Institute of Montreal, Montréal, Québec H2W 1R7, Canada.
J Biol Chem. 1999 Nov 26;274(48):34155-63. doi: 10.1074/jbc.274.48.34155.
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 beta-strands denoted A-G and A'-G', respectively. The alpha-chain (GMRalpha) 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 confers high affinity binding when associated with alpha (K(d) = 40-100 pM). 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 alpha-chain and residue Tyr(365) of the beta-chain, since mutations of either one of these residues resulted 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 residue of beta(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 GMRalpha and of beta(c), thus establishing a functional symmetry within an apparently asymmetrical heterodimeric structure.
粒细胞-巨噬细胞集落刺激因子（GM-CSF）受体（GMR）由两条链组成，它们属于以生长激素受体为代表的细胞因子受体超家族。细胞因子受体中常见的结构元件是由两个纤连蛋白样结构域组成的模块，每个结构域分别由七条β链表示为A-G和A'-G'。α链（GMRα）赋予GM-CSF低亲和力结合（K(d)=1-5 nM），而β链（β(c)）本身不结合GM-CSF，但与α链结合时赋予高亲和力结合（K(d)=40-100 pM）。在本研究中，我们通过多种方法的结合来确定配体识别和复合物稳定所需的分子决定因素，包括嵌合受体的构建、GM.GMR复合物三维模型的分子动力学以及定点诱变。然后通过平衡状态下的配体结合研究以及GM.GMR复合物动力学常数的测定来研究单个残基的功能重要性。对于这个三方复合物至关重要的是建立四个非共价键，其中三个决定了涉及α链的精氨酸（280）和酪氨酸（226）残基以及β链的酪氨酸（365）残基的配体识别过程的性质，因为这些残基中的任何一个发生突变都会导致结合速率显著降低。最后，β(c)的酪氨酸（365）残基具有双重功能，它与β(c)的另一个残基酪氨酸（421）协同作用以稳定复合物，因为酪氨酸（365）和酪氨酸（421）的突变会导致解离速率（Koff）急剧增加。有趣的是，这四个残基位于GMRα和β(c)的B'-C'和F'-G'环上，从而在明显不对称的异二聚体结构中建立了功能对称性。
