Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolincontaining fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment. INTRODUCTIONAcidic FGF (aFGF or FGF-1) belongs to the large FGF family of growth factors that play important roles in cell proliferation and differentiation (Burgess and Maciag, 1989;Crumley et al., 1991;Basilico and Moscatelli, 1992). The growth factors bind to transmembrane receptors with a cytoplasmic split tyrosine kinase domain. There are four identified FGF receptors (FGFRs) and a number of splicing variants (Johnson et al., 1990(Johnson et al., , 1991Hou et al., 1991;Partanen et al., 1991;Chellaiah et al., 1994). Different FGFs bind with different strengths to the different variants of the receptors. In addition, FGFs bind to cell surface heparans (Burgess and Maciag, 1989) as well as to a cysteine-rich binding protein of unknown function (Burrus et al., 1992;Gonatas et al., 1995).aFGF induces a strong mitogenic response in some cells, whereas in other cells it induces differentiation (Burgess and Maciag, 1989). It is not known what mechanism determines which response will be induced in each case. Upon binding of the growth factor to receptors, the tyrosine kinase is activated, apparently as a result of dimerization of the receptors by the bound growth factor (DiGabriele et al., 1998). Subsequently, a number of downstream signaling molecules are activated, such as phospholipase C␥ and MAPK (Mason, 1994). There is increasing evidence that externally added aFGF also acts intracellularly and that upon binding to the receptor the growth factor is able to penetrate cellular membranes and enter the cytosol and the nucleus (Imamura et al., 1990(Imamura et al., , 1994Zhan et al., 1992Zhan et al., , 1993Wiedlocha et al., 1994;Muñ oz et al., 1997;Klingenberg et al., 1998).After binding to FGFR, aFGF is internalized by endocytosis (Muñ oz et al., 1997). It has so far not been...
In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells. To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait. A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized. The insert contained an open reading frame corresponding to a novel protein of 42 kDa. The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence. In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF. FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency. Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes. The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF.
Three exciting papers in this issue of Developmental Cell provide new insights into the regulation of chondrocytic, osteoblastic, and osteoclastic differentiation during skeletal development and postnatal growth. The studies demonstrate that Wnt/beta-catenin signaling represents both a mechanism in mesenchymal precursor cells for selecting between chondrocytic and osteoblastic fates as well as a mechanism in osteoblasts for stimulating the production of an inhibitor of osteoclast formation.
The expression of several deletion mutants of fibroblast growth factor receptor 4 (FGFR4) was studied in COS-1 cells. FGFR4-mutants lacking most of the extracellular region did not efficiently reach the plasma membrane but accumulated in the endoplasmic reticulum (ER) and Golgi body. A mutant FGFR4 lacking the kinase domain as well as most of the extracellular region (ΔExt/R4Tth) had a distinct intracellular distribution. It localized in part to the nucleus, where it exhibited a striking spotted pattern. Ultrastructural studies showed that the nuclear spots consisted of several layers of membrane that were folded into onion-like structures at the nucleoplasmic side of the nuclear envelope. These intranuclear structures did not contain nuclear pores but were positive for the ER proteins calreticulin and protein disulfide isomerase, in addition to abundant ΔExt/R4Tth. Formation of the intranuclear structures was sensitive to inhibition of protein kinase C. Live microscopy of a green-fluorescent-protein/ΔExt/R4Tth fusion protein showed that the intranuclear structures were stable and immobile, suggesting that they function as deposits of the overexpressed mutant and associated membrane. The ΔExt/R4Tth protein also induced formation of densely packed membrane stacks in the cytosol and we suggest a model were the intranuclear structures are formed by invagination of ER-derived membrane stacks into the nucleus.
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