Bacterial Phytochrome as a Scaffold for Engineering of Receptor Tyrosine Kinases Controlled with Near-Infrared Light.

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.

More than the sum of the parts: Toward full-length receptor tyrosine kinase structures.

Intercellular communication governs complex physiological processes ranging from growth and development to the maintenance of cellular and organ homeostasis. In nearly all metazoans, receptor tyrosine kinases (RTKs) are central players in these diverse and fundamental signaling processes. Aberrant RTK signaling is at the root of many developmental diseases and cancers and it remains a key focus of targeted therapies, several of which have achieved considerable success in patients. These therapeutic advances in targeting RTKs have been propelled by numerous genetic, biochemical, and structural studies detailing the functions and molecular mechanisms of regulation and activation of RTKs. The latter in particular have proven to be instrumental for the development of new drugs, selective targeting of mutant forms of RTKs found in disease, and counteracting ensuing drug resistance. However, to this day, such studies have not yet yielded high-resolution structures of intact RTKs that encompass the extracellular and intracellular domains and the connecting membrane-spanning transmembrane domain. Technically challenging to obtain, these structures are instrumental to complete our understanding of the mechanisms by which RTKs are activated by extracellular ligands and of the effect of pathological mutations that do not directly reside in the catalytic sites of tyrosine kinase domains. In this review, we focus on the recent progress toward obtaining such structures and the insights already gained by structural studies of the subdomains of the receptors that belong to the epidermal growth factor receptor, insulin receptor, and platelet-derived growth factor receptor RTK families. © 2019 IUBMB Life, 71(6):706-720, 2019.

Synaptic and extrasynaptic location of the receptor tyrosine kinase met during postnatal development in the mouse neocortex and hippocampus.

MET, a replicated autism risk gene, encodes a pleiotropic receptor tyrosine kinase implicated in multiple cellular processes during development and following injury. Previous studies suggest that Met modulates excitatory synapse development in the neocortex and hippocampus, although the underlying mechanism is unknown. The peak of Met expression corresponds to the period of process outgrowth and synaptogenesis, with robust expression in hippocampal and neocortical neuropil. Resolving whether neuropil expression represents presynaptic, postsynaptic or glial localization provides insight into potential mechanisms of Met action. The subcellular distribution of Met was characterized using complementary ultrastructural, in situ proximity ligation assay (PLA), and biochemical approaches. At postnatal day (P) 7, immunoelectron microscopy revealed near-equivalent proportions of Met-immunoreactive pre- (axons and terminals) and postsynaptic (dendritic shafts and spines) profiles in the stratum radiatum in the hippocampal CA1 region. Staining was typically in elements in which the corresponding pre- or postsynaptic apposition was unlabeled. By P21, Met-immunoreactive presynaptic profiles predominated and ~20% of Met-expressing profiles were glial. A different distribution of Met-immunoreactive profiles was observed in layer V of somatosensory cortex: Met-labeled spines were rare and a smaller proportion of glial profiles expressed Met. Strikingly, Met-immunoreactive presynaptic profiles predominated over postsynaptic profiles as early as P7. PLA analysis of neurons in vitro and biochemical analysis of tissue subsynaptic fractions confirmed the localization of Met in specific synaptic subcompartments. The study demonstrates that Met is enriched at synapses during development and its activation may modulate synapse formation and stability through both pre- and postsynaptic mechanisms.

Prostaglandins differently regulate FGF-2 and FGF receptor expression and induce nuclear translocation in osteoblasts via MAPK kinase.

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF(2alpha) or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF(2alpha) stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF(2alpha). Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF(2alpha). Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF(2alpha) exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF(2alpha) stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.

Binding of discoidin domain receptor 2 to collagen I: an atomic force microscopy investigation.

Collagens have recently been identified as ligands for discoidin domain receptors (DDR1 and DDR2), generating an interest in studying the properties of binding of DDR to its ligand. We are interested in the interaction of DDR2 with collagen I because of its potential role in liver fibrosis. Our in vitro binding assay utilizes DDR2-Fc fusion proteins, which can be clustered (multimerized) by use of antibodies to form DDR2 complexes. Binding of DDR2 complexes to collagen I coated on plastic plates was established by a microplate-based assay using Eu(3+)-labeled proteins and time-resolved fluorometry. Clustering of the DDR2-Fc with antibody was found to be requisite for binding to collagen in vitro. Using atomic force microscopy (AFM) in an aqueous environment, we characterized the surface topographies of DDR2 complexes and collagen I, and investigated binding of this receptor-ligand pair. We were able to image and identify binding of DDR2 complexes onto individual molecules of triple-helical collagen and provide insight into the number and locations of binding sites on collagen I. In most cases, a single receptor complex bound to a single collagen molecule and there were preferred DDR2 binding sites on the collagen I triple helix. These data were validated by rotary-replication transmission electron microscopy (TEM) of glycerol-sprayed samples.

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA.

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.