The FGF21 Receptor Signaling Complex: Klothoß, FGFR1c, and Other Regulatory Interactions.

Scientific evidence is quickly growing that establishes FGF21 as a cytokine that signals both locally and systemically to induce metabolic effects. The focus of this chapter is the receptor/co-receptor signaling complex formed by endocrine FGF21. We provide an introduction to the major components of the complex including the Klotho family of co-receptors, fibroblast growth factor receptors (FGFRs), and the fibroblast growth factor ligands, placing each in the context of its own family members while emphasizing structural features that drive interaction. We subsequently focus specifically on FGF21 signaling through FGFR1c and KLB, describing what is known about each protein's structure and how this drives protein interaction and formation of the signaling complex at the plasma membrane. We subsequently explore the stoichiometry of FGFR1c and KLB at the plasma membrane before and after the addition of FGF21 ligand, comparing how unique features of the interaction could potentially affect signaling intensity. Finally, we discuss how formation of the signaling complex is potentially regulated by other regulatory interactions, including galectins, the extracellular matrix, and co-expression of FGFR5.

Bves directly interacts with GEFT, and controls cell shape and movement through regulation of Rac1/Cdc42 activity.

Bves is an integral membrane protein with no determined function and no homology to proteins outside of the Popdc family. It is widely expressed throughout development in myriad organisms. Here, we demonstrate an interaction between Bves and guanine nucleotide exchange factor T (GEFT), a GEF for Rho-family GTPases. This interaction represents the first identification of any protein that has a direct physical interaction with any member of the Popdc family. Bves and GEFT are shown to colocalize in adult skeletal muscle. We also demonstrate that exogenous expression of Bves reduces Rac1 and Cdc42 activity levels while not affecting levels of active RhoA. Consistent with a repression of Rac1 and Cdc42 activity, we show changes in speed of cell locomotion and cell roundness also result from exogenous expression of Bves. Modulation of Rho-family GTPase signaling by Bves would be highly consistent with previously described phenotypes occurring upon disruption of Bves function in a wide variety of model systems. Therefore, we propose Bves as a novel regulator of the Rac1 and Cdc42 signaling cascades.

Extracellular nucleotides act through P2U purinoceptors to elevate [Ca2+]i and enhance basic fibroblast growth factor-induced proliferation in sheep chondrocytes.

Extracellular nucleotides interact with specific cell surface receptors to mediate a variety of biological responses, including elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) in a number of cell types. Although extracellular ATP has been shown to affect chondrocyte function, the underlying mechanisms are poorly understood. In the present study, we investigated whether Ca2+-mobilizing purinoceptors are present on sheep chondrocytes. Chondrocytes were isolated from the proximal tibial growth plate of day 120-130 sheep fetuses. Early passage cells were loaded with indo-1 or fluo-3, and [Ca2+]i was monitored by fluorescence spectrophotometry. ATP (0.3-100 microM) induced transient elevation of [Ca2+]i, lasting approximately 1 min. Half-maximal elevation of [Ca2+]i was observed at an ATP concentration of 5.0 +/- 0.2 microM. Responses were still observed in the absence of extracellular Ca2+, and were abolished by pretreatment with thapsigargin, consistent with the release of Ca2+ from intracellular stores. Several nucleotides were tested for their ability to elevate [Ca2+]i. In order of potency, these were UTP approximately ATP >> ADP approximately 2-methylthio-ATP. No responses were elicited by benzoylbenzoic-ATP, a P2Z-selective agonist; alpha,beta-methylene-ATP, an agonist selective for certain P2X purinoceptors; AMP; adenosine; or pyrophosphate (all at 100 microM), demonstrating specificity. Taken together, these data indicate that nucleotides elevate [Ca2+]i in chondrocytes through interaction with the P2U purinoceptor subtype. Although pretreatment with pertussis toxin virtually abolished the Ca2+ response to lysophosphatidic acid, the response to UTP was relatively insensitive, suggesting that P2U purinoceptors are not linked to a pertussis toxin-sensitive G protein in chondrocytes. In contrast, the Ca2+ response to UTP was markedly inhibited by the biologically active phorbol ester 12-O-tetradecanoyl-beta-phorbol 13-acetate, but not by the inactive control compound 4 alpha-phorbol 12,13-didecanoate, suggesting that a 12-O-tetradecanoyl-beta-phorbol 13-acetate-sensitive isoform of protein kinase C regulates P2U purinoceptor signaling in these cells. UTP (10 microM) enhanced the proliferative response to basic fibroblast growth factor. The response to basic fibroblast growth factor was also enhanced by ATP, but not by 2-methylthio-ATP, consistent with involvement of P2U purinoceptors. Nucleotides released during trauma, inflammation, or cell death may act through P2U purinoceptors to regulate chondrocyte function in an autocrine or paracrine manner.

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 G1 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 contents were separated by differential centrifugation. FGFBPs were separated using FGF-2 affinity chromatography. Ligand blot analysis using 125I-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 G1, 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 G1. Immunoprecipitation was performed to monitor newly synthesized FGFR1 migration throughout the cell cycle. Synchronized cells were cultured in medium containing 35S-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 G1 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 G1, suggesting that this was not derived from the plasma membrane. The truncated FGFR1 may mediate the nuclear translocation of FGF-2 during late G1.