Mutation of the DRY motif in Cb2 and analysis of 32D/G-CSF-R/Cb2 mutant clones. (A) Location of the DRY motif in Cb2. Introduced mutations are indicated in bold. The box shows the results of ligand binding assays. The dissociation constant (Kd) is expressed for CP55940 in picomolars and for 2-AG in nanomolars. Maximum binding (Bmax) is expressed as femtomoles per milligram of protein. Data were pooled from independent experiments performed on 2 clones of the same cell type. (B) Flow cytometric analysis of representative 32D/G-CSF-R clones expressing the distinct constructs. The insets show cell fluorescence distribution in the infected cells by microscopy; original magnification × 63. (C) Four representative mutant clones cultured for 8 days in the presence of G-CSF with or without CP (100 nM). (D) Four representative 32D/G-CSF-R clones expressing Cb2 mutants cultured in G-CSF with or without CP, Cb2 (C2), and Cb1 (C1) inverse agonist (100 nM). Counts were carried out on day 8 of culture. □ indicates blast cells; ▪, intermediate forms; and ▦, terminally differentiated neutrophils. (E) In vitro migration of cells containing a DRY, DRA, or DAY motif. Cells were exposed to medium with 300 nM 2-AG or control medium; 100 nM C1 or C2 was added to the upper chamber.The percentage of migration is the average of 3 clones.
