IL-2 is a cytokine that functions as a growth factor and central regulator in the immune system and mediates its effects through ligand-induced hetero-trimerization of the receptor subunits IL-2R␣, IL-2R␤, and ␥c. Here, we describe the crystal structure of the trimeric assembly of the human IL-2 receptor ectodomains in complex with IL-2 at 3.0 Å resolution. The quaternary structure is consistent with a stepwise assembly from IL-2͞IL-2R␣ to IL-2͞IL-2R␣͞IL-2R␤ to IL-2͞IL-2R␣͞IL-2R␤͞␥c. The IL-2R␣ subunit forms the largest of the three IL-2͞IL-2R interfaces, which, together with the high abundance of charge-charge interactions, correlates well with the rapid association rate and high-affinity interaction of IL-2R␣ with IL-2 at the cell surface. Surprisingly, IL-2R␣ makes no contacts with IL-2R␤ or ␥c, and only minor changes are observed in the IL-2 structure in response to receptor binding. These findings support the principal role of IL-2R␣ to deliver IL-2 to the signaling complex and act as regulator of signal transduction. Cooperativity in assembly of the final quaternary complex is easily explained by the extraordinarily extensive set of interfaces found within the fully assembled IL-2 signaling complex, which nearly span the entire length of the IL-2R␤ and ␥c subunits. Helix A of IL-2 wedges tightly between IL-2R␤ and ␥c to form a three-way junction that coalesces into a composite binding site for the final ␥c recruitment. The IL-2͞␥c interface itself exhibits the smallest buried surface and the fewest hydrogen bonds in the complex, which is consistent with its promiscuous use in other cytokine receptor complexes.common ␥ chain ͉ cooperativity ͉ IL-2 receptor ͉ receptor assembly ͉ structure-activity relationship

Fibroblast growth factors (FGFs) effect cellular responses by binding to FGF receptors (FGFRs). FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation. We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2. The crystal structure, determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein, is a dimeric assemblage of 1:1 ligand: receptor complexes. FGF is bound at the junction between the two domains of one FGFR, and two such units are associated through receptor:receptor and secondary ligand:receptor interfaces. Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains. This dimeric assemblage provides a structural mechanism for FGF signal transduction. Fibroblast growth factors (FGFs) stimulate a variety of cellular functions by binding to cell surface FGF receptors (FGFRs) in the presence of heparin proteoglycans. FGFRs are single-chain receptor tyrosine kinases that become activated through autophosphorylation that is thought to be induced through a mechanism of ligand-mediated receptor oligomerization (1). Receptor activation gives rise to a signal transduction cascade that leads to gene activation and diverse biological responses (2, 3). Both FGFs and FGFRs are expressed in defined spatial and temporal patterns, and they are involved in differentiation of both epithelial and neuronal cells. FGFs are potent mitogens for many cell types. Aberrant signaling through FGFR can lead to tumorigenesis and skeletal disorders (4).The FGF system has appreciable diversity in both ligands and receptors. The FGF family contains at least 15 distinct factors, highly conserved across mammalian species but divergent (30-70% sequence identity) among paralogs (2). The FGFR family includes four identified genes and numerous subtypes of alternatively spliced isoforms, particularly within the well characterized FGFR1 and FGFR2 types. Differential responses follow from this diversity.FGFRs have an extracellular portion imbued with the ligandbinding potential, a transmembrane segment, and a tyrosine kinase domain in the cytoplasm (2). The extracellular portion comprises three Ig-like domains, D1, D2, and D3, with an acidic stretch of approximately 30 residues, the acid box, between D1 and D2. Isoforms generated by alternate splicing events include receptors that lack Ig-like domain D1 or both D1 and the acid box, as well as variants having two alternative sequences for the C-terminal half of the third Ig-like domain. The FGF binding site has been mapped to domains D2, D3, and the interdomain linker.FGFs are secreted factors originally identified based on their mitogenicity toward fibroblasts. They are small proteins for which several FGF crystal structures have been determined; all have 12 ␤ strands in a ␤-trefoil fold (2). Mutational analyses have mapped the sites of interaction...

The biological activity of the transcription factor NF-B is differentially controlled by three IB proteins, IB␣, IB␤, and IB. We have examined the molecular basis for the differential inhibitory strengths of IB proteins by constructing hybrid IBs and found that the first ankyrin repeat of IB␣ is responsible for its strong inhibitory effect. Swapping a putative ␤-turn within the first ankyrin repeat between the strong IB␣ and the weak IB␤ inhibitors switches their in vivo inhibitory activity on NF-B. The qualitatively distinct contacts made by this ␤-turn in IB␣ and IB␤ with NF-B determine the efficiency by which IBs sequester NF-B to the cytoplasm, thus explaining their distinct effects on gene activity.The transcription factor NF-B orchestrates the activation of numerous genes involved in the control of cell activities in the immune system and is also vital for craniofacial, liver, and limb development in higher eukaryotic organisms. NF-B exists in virtually all cell types in the form of dimeric complexes consisting of different members of the Rel family of proteins. In mammals, there are five Rel proteins, p50, p52, p65, c-Rel, and RelB, all of which share an amino-terminal 300 amino acid conserved region known as Rel Homology Region. This region is responsible for DNA binding, dimerization, and nuclear localization. Unlike most transcriptional activators, NF-B resides in the cytoplasm and must therefore translocate to the nucleus to function. Association with the inhibitory IB proteins tightly regulates the activity of NF-B. These interactions have two functional consequences. First, NF-B͞IB complexes are sequestered in the cytoplasm, because IBs mask the nuclear localization signal (NLS) of NF-B, presumably by means of direct protein-protein interactions, and secondly, IBs can inhibit NF-B DNA binding. In response to a large variety of extracellular stimuli, the IB proteins, while still bound to NF-B, are phosphorylated, ubiquitinated, and finally degraded by the proteasome. The free NF-B translocates to the nucleus, where it activates gene transcription (reviewed in refs. 1-5).The IB family consists of three members, IB␣, IB␤, and IB (6-10). Importantly, the carboxyl-terminal regions of the precursors for p50 and p52, p105, and p100, respectively, can also function as IBs. Each member of the family contains six copies of a 33 amino acid module known as ankyrin repeat, which functions as a protein-protein interaction domain. The region carboxyl-terminal to the ankyrin repeats contains a proline (P), glutamate (E), serine (S), and threonine (T) (PEST) sequence regulating basal level protein turnover and is also required for inhibition of DNA binding, whereas the amino-terminal region is the signal responsive domain (2, 5). Despite their extensive structural similarities, IB␣, IB␤, and IB exhibit substantial differences in vivo (10-13). Depending on the cell type and on the stimulus, IBs respond differentially to NF-B-inducing signals. In general, IB␣ is rapidly degraded, whereas IB␤ and IB are degrade...

Cell interaction with extracellular matrix (ECM) modulates cell growth and differentiation. By using in vitro culture systems, we tested the effect of type I collagen (Coll-I) on signal transduction mechanisms in the osteosarcoma cell line UMR-106 and in primary cultures from neonatal rat calvariae. Cells were cultured for 72 h on Coll-I gel matrix and compared with control cells plated on plastic surfaces. Agonist-dependent and voltage-dependent rises in cytosolic Ca2+ concentration ([Ca2+]i; measured by fura 2 fluorometry) were significantly blunted in cells cultured on Coll-I compared with cells grown on plastic. In UMR-106 cells, the collagen matrix effect was mimicked by 24-h incubation with soluble Coll-I or short peptides containing the arginine-glycine-aspartate motif. Accumulation of cellular adenosine 3',5'-cyclic monophosphate (cAMP) stimulated by parathyroid hormone, cholera toxin, and forskolin was augmented (50-150%) in cells plated on Coll-I vs. control. The collagen effect on both [Ca2+]i- and adenylate cyclase-signaling pathways in UMR-106 cells was abrogated in the presence of protein kinase C (PKC) depletion or inhibition. Also, Coll-I induced a twofold increase in membrane-bound PKC without changing cytosolic PKC activity. Thus, by altering PKC activity, Coll-I modulates the [Ca2+]i- and cAMP-signaling pathways in osteoblasts. This, in turn, may influence bone remodeling processes.

(a) Phosphate depletion induced hypercalcemia and hypercalciuria that were not reduced by APD administration. (b) The calcemic response to PTH was reduced in phosphate-depleted rats and was unaffected by APD administration in normal and phosphate-depleted rats, and (c) APD administration markedly changed bone histology without affecting the biochemical changes induced by phosphate depletion.

No abstract

No abstract

No abstract