Perinuclear localization of an intracellular binding protein related to the fibroblast growth factor (FGF) receptor 1 is temporally associated with the nuclear trafficking of FGF-2 in proliferating epiphyseal growth plate chondrocytes

Fibroblast growth factor-2 (FGF-2) is a potent autocrine mitogen for fetal epiphyseal growth plate chondrocytes and exhibits a transient nuclear translocation during G(1) of the cell cycle. We have characterized an intracellular binding protein (FGFBP) for FGF-2 that undergoes a juxtanuclear localization coincident with the nuclear translocation of the growth factor. Chondrocytes were isolated from the proliferative zone of the ovine fetal proximal tibial growth plate at 50-130 days gestation by collagenase digestion and were maintained in monolayer at early passage number. Cells were growth restricted by serum starvation for 48 h, and the synchronized culture was restarted into the cell cycle in the presence of 2% FBS. Cells were removed between 4-26 h of incubation, and fractions representing the plasma membrane, cytoplasm, nuclear membrane, and nuclear con tents were separated by differential centrifugation. FGFBPs were separated using FGF-2 affinity chromatography. Ligand blot analysis using I-125-labeled FGF-2 showed that a FGFBP of 46-48 kDa (represented by a double band) was present on the nuclear membrane at mid to late G(1), and Western blot showed this to be immunologically related to a part of the extracellular domain of the high affinity FGF receptor 1 (FGFR1). Immunocytochemistry with intact cell cultures showed that this protein underwent a juxtanuclear distribution through mid to late G(1). Immunoprecipitation was performed to monitor newly synthesized FGFR1 migration throughout the cell cycle. Synchronized cells were cultured in medium containing S-35-labeled methionine/cysteine, and the cellular compartments were separated before immunoprecipitation using an antibody raised against the extracellular domain of FGFR1. Newly synthesized FGFR1-related proteins appeared throughout G(1) and migrated multidirectionally within the cell; intact receptor of 125-145 kDa accumulated at the plasma membrane, while both intact receptor and truncated FGFR1 of 46-48 kDa were detected on the nuclear membrane, but not within the nucleus. Cells were incubated with protamine sulfate to prevent the binding of endogenous, cell membrane-associated FGF-2 to high affinity FGFRs and their subsequent internalization. This did not alter the juxtanuclear accumulation of truncated FGFR1 in late G(1), suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation of FGF-2 during late G(1).