Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor - Importance of a glutamate residue in the transmembrane region

To analyze the function of each subunit of the receptor for granulocyte-mac rophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmemb rane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). me demonstrated the capacity of alpha/be ta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent acti vation of tyrosine kinase activity and proliferation in transfected Ba/F3 c ells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphologi cal changes, expression of alpha(4)- and beta(1)-integrin receptor subunits , and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point muta nts changing this amino acid and its position were expressed in Ba/F3 cells . None of these mutants was capable of supporting GM-CSF-dependent prolifer ation; however, when beta-GMR was coexpressed, GM-CSF mediated short and lo ng term proliferation. Interestingly, some mutants but not alpha/beta-GMR c an induce proliferation in the presence of an anti-alpha-GMR antibody. Thes e data demonstrate the significance of a glutamate residue in the transmemb rane region of alpha/beta-GMR for ligand-induced receptor activation.