PMA- and LPS-induced CSF-1 receptor down-modulation results in the appearance of a 45-kDa receptor fragment. (A) p388D1 macrophages were stimulated with 100 ng of PMA/ml for the indicated amounts of time. CSF-1 receptors were isolated by immunoprecipitation and analyzed by anti-CSF-1 receptor immunoblotting. (B) 293 cells were transfected with a CSF-1 receptor expression construct (lanes 1 to 10) or with a control plasmid (lane 11). The cells were stimulated with PMA for the indicated amounts of time and analyzed as described for panel A. (C) p388D1 macrophages were stimulated with 100 ng of LPS/ml for the indicated amounts of time and analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting. (D) p388D1 macrophages were stimulated with 100 ng of CSF-1 or PMA/ml for the indicated amounts of time and analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting. CD is the CSF-1 receptor cytoplasmic domain.

The CSF-1 receptor cytoplasmic domain is degraded in the proteosome. Control p388D1 macrophages (lanes 1 to 3) and macrophages pretreated with the proteosomal inhibitor LL-n-L (lanes 4 to 6) or the lysosomal inhibitor methylamine (lanes 7 to 9) were left unstimulated (lanes 1, 4, and 7) or were stimulated with PMA for 15 min (lanes 2, 5, and 8) or for 1.5 h (lanes 3, 6, and 9). The cells were lysed and analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting.

The CSF-1 receptor transmembrane domain directs degradation of the insulin receptor cytoplasmic domain. 293 cells were transfected with expression constructs for the wild-type insulin receptor (IR; lanes 1 to 3), the CSF-1R/IR_tm-cyto chimera (lanes 4 to 6), the CSF-1R/IR_cyto chimera (lanes 7 to 9), or a control plasmid (lane 10). Cells were left untreated (lanes 1, 4, and 7) or were stimulated with PMA for 15 min (lanes 2, 5, 8, and 10) or for 1.5 h (lanes 3, 6, and 9). The cells were lysed and analyzed by immunoprecipitation and immunoblotting with an antiserum against the insulin receptor β chain.

Dominant-negative PS1 and a γ-secretase inhibitor interfere with the release of the CSF-1 receptor cytoplasmic domain into the cytosol. (A) 293 cells were transfected with expression constructs for the wild-type CSF-1 receptor (lanes 1 to 6 and 8 to 13) or with a control plasmid (lanes 7 and 14) and an expression construct for dominant-negative PS1 (lanes 4 to 7 and lanes 11 to 14) or a control plasmid (lanes 1 to 3 and lanes 8 to 10). Control cells (lanes 1, 4, 8, and 11) or cell stimulated with PMA for 15 min (lanes 2, 5, 7, 9, 12, and 14) or for 1.5 h (lanes 3, 6, 10, and 13) were lysed and separated into a particulate fraction (lanes 1 to 7) and a soluble fraction (lanes 8 to 14). Both fractions were analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting (top panel). Whole-cell lysates were analyzed by anti-PS1 immunoblotting (bottom panel). (B) Control p388D1 macrophages (lanes 1 to 3 and 7 to 9) or macrophages pretreated with the γ-secretase inhibitor L-685,458 (lanes 4 to 6 and 10 to 12) were left unstimulated (lanes 1, 4, 7, and 10) or were stimulated with PMA for 15 min (lanes 2, 5, 8, and 11) or for 1.5 h (lanes 3, 6, 9, and 12). The cells were lysed and separated into a particulate (lanes 1 to 6) and a soluble (lanes 7 to 12) fraction. Both fractions were analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting.

Alignment of transmembrane domains of γ-secretase substrates. The transmembrane domains of several γ-secretase substrates were aligned (2, 20, 30, 35, 43, 44, 47, 50, 51, 69). The transmembrane domain of the insulin receptor was included for comparison. Conserved residues are boxed, and stop-transfer signals are shown in grey.

Release of the CSF-1 receptor cytoplasmic domain into the cytoplasm following activation of PKC. Control macrophages (lanes 1 and 4) or macrophages stimulated with PMA for 15 min (lanes 2 and 5) or for 1.5 h (lanes 3 and 6) were lysed and separated into a particulate fraction (lanes 1 to 3) and a soluble fraction (lanes 4 to 6). Both fractions were analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting.

Down-modulation of a CSF-1R/IR chimera. (A) Domain swapping was carried out using the wild-type murine CSF-1 receptor (CSF-1R) and the human insulin receptor (IR). CSF-1R/IR_tm-cyto contains the extracellular domain of the CSF-1 receptor and the transmembrane and intracellular region of the insulin receptor. CSF-1R/IR_cyto contains the extracellular and the transmembrane domains of the CSF-1 receptor and the intracellular region of the insulin receptor. In CSF-1R/IR_tm the CSF-1 receptor transmembrane domain has been replaced with the insulin receptor transmembrane domain. In CSF-1R/IR_jm the CSF-1 receptor juxtamembrane region has been replaced with the insulin receptorjuxtamembrane region. In CSF-1R/IR_kd the CSF-1 receptor kinase domain has been replaced with the insulin receptor kinase domain. tm = transmembrane domain; jm = juxtamembrane region; kd = kinase domain; ki = kinase insert; ct = carboxy-terminal region. (B) 293 cells were transfected with expression constructs for the wild-type insulin receptor (IR; lanes 1 to 3) or the CSF-1R/IR_tm-cyto chimera (lanes 4 to 7), or with a control plasmid (lane 7). Cells were left untreated (lanes 1 and 4) or were stimulated with PMA for 15 min (lanes 2, 5, and 7) or for 1.5 h (lanes 3 and 6). The cells were lysed and analyzed by immunoprecipitation and immunoblotting with an antiserum against the insulin receptor β chain. (C) 293 cells were transiently transfected with expression constructs for the wild-type CSF-1 receptor (lanes 1 to 3), the CSF-1R/IR_tm chimera (lanes 4 to 6), the CSF-1R/IR_jm chimera (lanes 7 to 9), the Δct CSF-1 receptor mutant (lanes 10 to 12), or a control plasmid (lane 13). Control cells (lanes 1, 4, 7, and 10) and cells treated with PMA for 15 min (lanes 2, 5, 8, 11, and 13) or 1.5 h (lanes 3, 6, 9, and 12) were lysed and analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting. (D) 293 cells were transiently transfected with expression constructs for the wild-type CSF-1 receptor (lanes 1 to 3), the Δki CSF-1 receptor mutant (lanes 4 to 6), the CSF-1R/IR_kd chimera (lanes 7 to 9), or a control plasmid (lane 10). Control cells (lanes 1, 4, and 7) and cells stimulated with PMA for 15 min (lanes 2, 5, 8, and 10) or for 1.5 h (lanes 3, 6, and 9) were lysed and analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting.

The transmembrane domain is essential for release of the CSF-1 receptor cytoplasmic domain into the cytosol. 293 cells were transfected with expression constructs for the wild-type CSF-1 receptor (lanes 1 to 3 and 8 to 10), the CSF-1R/IR_tm chimera (lanes 4 to 6 and 11 to 13), or a control plasmid (lanes 7 and 14). Control cells (lanes 1, 4, 8, and 11) or cells stimulated with PMA for 15 min (lanes 2, 5, 7, 9, 12, and 14) or for 1.5 h (lanes 3, 6, 10, and 13) were lysed and separated into a particulate fraction (lanes 1 to 7) and a soluble fraction (lanes 8 to 14). Both fractions were analyzed by anti-CSF-1 receptor immunoprecipitation and immunoblotting.

CSF-1 receptors are processed at the plasma membrane. Control p388D1 macrophages and macrophages pretreated with the γ-secretase inhibitor L-685,458 were left unstimulated or were stimulated with PMA for 10 or 90 min and analyzed by anti-CSF-1 receptor immunofluorescence.

Model for CSF-1 receptor down-modulation and release of its cytoplasmic domain. PMA-induced activation of PKC leads to cleavage of the CSF-1 receptor ectodomain by TACE. TACE recognition of the CSF-1 receptor is only dependent upon extracellular sequences. The remaining membrane-bound cytoplasmic domain (CD) is recognized by γ-secretase and cleaved within the transmembrane domain. Intramembrane cleavage is dependent on the CSF-1 receptor transmembrane sequence and results in the release of the CD into the cytosol. Once released into the cell, the CD localizes in part to the nucleus and is degraded by the proteosome.