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 f etal epiphyseal growth plate chondrocytes and exhibits a transient nuc lear translocation during G(1) of the cell cycle. We have characterize d 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 ge station by collagenase digestion and were maintained in monolayer at e arly passage number. Cells were growth restricted by serum starvation for 48 h, and the synchronized culture was restarted into the cell cyc le in the presence of 2% FBS. Cells were removed between 4-26 h of inc ubation, and fractions representing the plasma membrane, cytoplasm, nu clear membrane, and nuclear con tents were separated by differential c entrifugation. FGFBPs were separated using FGF-2 affinity chromatograp hy. 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 nuclea r membrane at mid to late G(1), and Western blot showed this to be imm unologically 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 distributi on through mid to late G(1). Immunoprecipitation was performed to moni tor newly synthesized FGFR1 migration throughout the cell cycle. Synch ronized cells were cultured in medium containing S-35-labeled methioni ne/cysteine, and the cellular compartments were separated before immun oprecipitation using an antibody raised against the extracellular doma in of FGFR1. Newly synthesized FGFR1-related proteins appeared through out G(1) and migrated multidirectionally within the cell; intact recep tor of 125-145 kDa accumulated at the plasma membrane, while both inta ct receptor and truncated FGFR1 of 46-48 kDa were detected on the nucl ear membrane, but not within the nucleus. Cells were incubated with pr otamine sulfate to prevent the binding of endogenous, cell membrane-as sociated FGF-2 to high affinity FGFRs and their subsequent internaliza tion. This did not alter the juxtanuclear accumulation of truncated FG FR1 in late G(1), suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation o f FGF-2 during late G(1).