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-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane 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). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological 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 mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.
Kafert
S
S
Department of Hematology, Hannover Medical School, D-30625 Hannover, Germany.
Luther
S
S
Böll
I
I
Wagner
K
K
Ganser
A
A
Eder
M
M
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D048429:Cell Size / Q000235:genetics; D005434:Flow Cytometry; D018698:Glutamic Acid / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D016023:Integrins / Q000378:metabolism; D007377:Interleukin-3 / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
D000906:Antibodies; D016023:Integrins; D007377:Interleukin-3; D008565:Membrane Proteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017346:Protein Serine-Threonine Kinases