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Regulation of Receptor Binding Specificity of FGF9 by an Autoinhibitory Homodimerization.

ABSTRACT: The epithelial fibroblast growth factor 9 (FGF9) subfamily specifically binds and activates the mesenchymal "c" splice isoform of FGF receptors 1-3 (FGFR1-3) to regulate organogenesis and tissue homeostasis. The unique N and C termini of FGF9 subfamily ligands mediate a reversible homodimerization that occludes major receptor binding sites within the ligand core region. Here we provide compelling X-ray crystallographic, biophysical, and biochemical data showing that homodimerization controls receptor binding specificity of the FGF9 subfamily by keeping the concentration of active FGF9 monomers at a level, which is sufficient for a normal FGFR "c" isoform binding/signaling, but is insufficient for an illegitimate FGFR "b" isoform binding/signaling. We show that deletion of the N terminus or alanine substitutions in the C terminus of FGF9 skews the delicate ligand equilibrium toward active FGF9 monomers causing off-target binding and activation of FGFR b isoforms. Our study is the first to implicate ligand homodimerization in the regulation of ligand-receptor specificity.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5587394 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Regulation of Receptor Binding Specificity of FGF9 by an Autoinhibitory Homodimerization.

Liu Yang Y Ma Jinghong J Beenken Andrew A Srinivasan Lakshmi L Eliseenkova Anna V AV Mohammadi Moosa M

Structure (London, England : 1993) 20170727 9

The epithelial fibroblast growth factor 9 (FGF9) subfamily specifically binds and activates the mesenchymal "c" splice isoform of FGF receptors 1-3 (FGFR1-3) to regulate organogenesis and tissue homeostasis. The unique N and C termini of FGF9 subfamily ligands mediate a reversible homodimerization that occludes major receptor binding sites within the ligand core region. Here we provide compelling X-ray crystallographic, biophysical, and biochemical data showing that homodimerization controls rec ...[more]

PMID: 28757146

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Project description:BackgroundHuman influenza viruses are known to bind to sialic acid linked alpha2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection.ResultsViruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of alpha2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for alpha2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of alpha2-6 and alpha2-3 sialic acids, including human A549 and HeLa cells with high levels of alpha2-6 sialic acid, and CHO cells that have only alpha2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells.ConclusionAgglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of alpha2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of alpha2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with alpha2-6 receptor specificity.

Project description:Increasing evidence suggests that G protein-coupled receptors form functional dimers or larger oligomeric complexes through homo- or heterodimerization, and that various transmembrane (TM) domains contribute dimerization interfaces. In this study, monomeric receptor structures - either the monomeric crystallographic structure of bovine rhodopsin or an A(3) adenosine receptor (AR) homology model - were dimerized by computational methods assuming various TM contact regions, optimized, and compared. The semi-empirical oligomeric structure of mouse rhodopsin studied in a native disc membrane with atomic force microscopy was used to establish the distance between monomers in the initial dimeric models. Among eight variations of symmetrical homodimers of bovine rhodopsin, the favored dimeric assembly closely resembled the semi-empirical model, in which TM domains 4 and 5 were the contact site, thus validating this approach. We used similar methods to generate eight homodimers of the A(3)AR and found the favored dimeric interface similarly to be TM4-5. By this method, dimeric variations - TM1-2, TM2-3, TM2-4, TM3-4, TM4-5, TM5-6, TM6-7, and TM7-1 - were constructed with the SYBYL 7.0 program by using a novel "fit-centroids-normal" method. Fitting atoms considered one of eight TM-TM centroids or seven-TM centroids, two centroids of each monomer, and a normal atom passing through the plane containing all centroids. Following molecular dynamics, the most energetically favorable contact modes were identified. In addition to TM4-5, which was favored in both rhodopsin and A(3)AR dimeric models, TM1-2 dimers in which helices 8 also contacted each other were judged favorable. The largest contact surface area between the monomers among the various homodimers, determined by van der Waals calculation with the MOLCAD surface program, was for the TM4-5 dimer. This contact surface also showed a high degree of shape complementarity. In addition, the TM4-5 dimers made by this theoretical method were more stable than the semi-empirically determined dimer.

Dataset Information

Regulation of Receptor Binding Specificity of FGF9 by an Autoinhibitory Homodimerization.

Publications

Regulation of Receptor Binding Specificity of FGF9 by an Autoinhibitory Homodimerization.

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