Lubert Stryer - US grants

The overall goal of this research is to elucidate the molecular basis of visual excitation and adaptation in retinal rod cells. We plan to carry out the following enzymatic, spectroscopic, structural, and electrophysiological studies of rod outer segment proteins: (1) The light-triggered amplification cycle in rod outer segments involving photoexcited rhodopsin, transducin, and the cyclic GMP phosphodiesterase (PDE) will be investigated in molecular detail. Fluorescence energy transfer studies will be carried out to establish the location of the subunits of transducin relative to each other, R*, and the disk membrane. (2) The structural differences between membrane-bound and soluble GTP- transducin will be determined and the functional significance of the two forms will be ascertained by reconstitution experiments. (2) Energy transfer studies will also be carried out to determine how activated transducin reverses the inhibition of PDE imposed by its gamma subunit. Specifically, does transducin displace the gamma subunit from the inhibited holoenzyme or does it carry it away? (3) A synthetic gene for the gamma subunit has been prepared for site-specific mutagenesis studies. The aim is to pinpoint the region of gamma that binds to the catalytic subunits and blocks their activity It will also be interesting to engineer mutants that irreversibly inhibit PDE. (4) Nonhydrolyzable analogs of cGMP will be used to determine where most of the cGMP in rod outer segments is bound and how its uptake and release are controlled. Defects of cGMP buffer sites may be important in the pathogenesis of some degenerative diseases of the retina. (5) We have recently found that guanylate cyclase is activated by small decreases in the concentration of Ca2+ in the vicinity of 10-7 M. The control of guanylate cyclase by Ca2+ is likely to be important for recovery and adaptation. The calcium-binding protein mediating this highly cooperative effect will be purified and its mechanism of regulation will be investigated. (6) The molecular architecture of the cGMP- activated channel in the plasma membrane will be probed by fluorescent analogs of cGMP. Energy transfer will be used as a spectroscopic ruler to map the channel. Fluorescence and conductance studies of reconstituted membranes containing purified channel protein will define the allosteric mechanism by which cGMP cooperatively opens the conductance pathway. The conductance properties of channels containing a defined number of covalently attached cGMP analog molecules should be highly informative in revealing how cGMP allosterically opens the channel. A detailed understanding of the cyclic GMP cascade of vision will be highly rewarding in understanding signal transduction processes generally and molecular diseases arising from defective coupling.

The overall aims are to develop novel fluorescence techniques and use them in concert with other experimental approaches to elucidate the structure and dynamics of selected proteins in the next five years, we will focus on (1) the molecular mechanism of triggering of effector functions of immunoglobulins, (2) the functional significance of segmental flexibility, and (3) the development of new phycobiliprotein fluorescent probes for use in high-sensitivity fluorescence assays. Conformational transitions induced by the binding of antigen will be detected by fluorescence studies of probes attached to specific sites in each of the immunoglobulin domains and the hinge region. Reactive cysteine residues will be introduced by site-specific mutagenesis of serine and alanine residues near the surface. Fluorescent probes will be attached to these cysteines. The effects of binding univalent and multivalent antigens will be monitored by changes in the emission spectrum, quantum yield, and excited-state lifetime of the fluorescent probes. Distances between pairs of specifically labeled sites will be determined using fluorescence energy transfer as a spectroscopic ruler. Diffusion-enhanced energy transfer using long-lived terbium chelates will provide information concerning the depth and accessibility of labeled cysteine residues. Rotation motions of domains will be delineated by nanosecond fluorescence polarization spectroscopy. The hinge region and adjacent residues in the CH1 domain will be changed by site-specific mutagenesis to gain insight into the control of segmental flexibility and its functional significance. These studies should reveal whether complement fixation is triggered by the clustering of Fc units, the unmasking of effector sites by movements of domains, or by propagated conformational changes that activate the effector site. The effect of antigen on the conformation and dynamics of membrane-bound immunoglobulin in reconstituted membranes will also be investigated by fluorescence techniques to gain insight into transmembrane signaling. New phycobiliprotein fluorescent probes that emit in the far red and near infrared will be synthesized for use in multiparameter fluorescence-activated cell sorting and high-sensitivity fluorescence immunoassays. Phycobiliproteins with energy acceptors joined to them by a cleavable bond will be prepared as substrates for enzyme-linked immunoassays. A microfluorimeter optimized for detecting particles labeled with phycofluors (e.g., microorganisms) will be constructed.

Many cells exhibit calcium spikes (periodic transient increases in cytosolic CA2+) when stimulated by a neurotransmitter, hormone, or growth factor. The overall goal of this research is to elucidate the molecular mechanism of calcium spiking and delineate its role in signal transduction. The rat basophilic leukemic cell (RBL cell) will be studied as a model secretory cell, the rat hepatocyte as an integrator of diverse stimuli, and the PC12 cell as a model neuron. Calcium spiking will be monitored by photon-counting fluorescence microscopy of single cells containing an indicator such as fluo-3. Biochemical, fluorescence, and ultrastructural studies of intact cells, permeabilized cells, purified proteins, and reconstituted membrane assemblies will be carried out to answer: (1) How are calcium spikes generated? We will test a molecular model for calcium spiking that is based on four elements: cooperativity and positive feedback between inositol 1,4,5- trisphosphate (IP3) and cytosolic Ca2+, delayed deactivation by mitochondrial uptake of Ca2+, and reactivation by refilling of the endoplasmic reticulum Ca2+ store. Specific inhibitors will pinpoint the contributions of particular processes to spike generation. (2) How is calcium spiking modulated? The effects of calcium influx into the cell, intracellular pH, the level of phosphoinositides, and phosphorylation state of components of the spike generator will be determined. (3) How is spiking altered by the interplay of the phosphoinositide cascade with other signal transduction pathways? The modulatory actions of the cyclic AmP cascade, growth factors, voltage-sensitive calcium channels, and lithium ion will be investigated. (4) How do calcium spikes trigger effector events such as exocytosis and memory? The distribution of F- actin and myosin in stimulated RBL cells will be determined by fluorescence and immunoelectron microscopy to determine whether individual spikes induce discrete cytoskeletal rearrangements and to relate them to granule release. The Ca2+/calmodulin-dependent protein kinase of PC12 cells will be studied as a model memory protein. We will measure how calcium spikes switch this protein between different functional states as expressed by their degree of phosphorylation- autophosphorylation activity, and kinase activity for exogenous substrates such as tyrosine hydroxylase. A deeper understanding of calcium spiking is likely to reveal how digital logic is used within cells to process information and achieve timing control (as in circadian rhythms). Some neuropsychiatric disorders may arise from kinetic mismatches of components of the spike generator. Information about spiking should lead to a better understanding of the therapeutic action of lithium ion in manic-depressive disorders.

The structure and mechanism of action of a family of myristoylated calcium-sensing proteins, exemplified by retinal recoverin, will be investigated by fluorescence, NMR, and spin-label spectroscopy. These acylated proteins move from the cytosol to a membrane on binding calcium. The aim is to determine the molecular mechanism of calcium-myristoyl switches and define their roles in signal transduction processes. Having solved the three-dimensional structures of calcium-free myristoylated recoverin and calcium-bound unmyristoylated recoverin, we turn now to the determination of the structure of the calcium-bound form of myristoylated recoverin to reveal the precise structural basis of the switch. Our working hypothesis is that recoverin and brain homologs such as neurocalcin and hippocalcin serve to couple calcium cascades to G-protein cascades by interacting with kinases that deactivate seven-helix receptors. The membrane-contact sites of recoverin in the Ca2+-bound state and their depth will be determined by spin-label ESR spectroscopy using nitroxide-labeled myristate and nitroxide-tagged cysteines introduced by mutagenesis. Structural, spectroscopic, and functional studies of the interaction of Ca2+-bound recoverin with rhodopsin kinase and fragments of this target will also be carried out. Three classes of recoverin mutants will be generated and analyzed to further our understanding of the structure and dynamics of this sensor: (1) Nonpolar residues in the myristoyl binding pocket will be replaced by polar ones. (2) Glycines in putative hinge regions will be changed to alanine. (3) Charged residues surrounding a concave hydrophobic surface, the putative target-binding site, will be charged to oppositely-charged residues. Structural studies of neurocalcin and hippocalcin will also be carried out and targets of these brain homologs will be identified using the yeast two-hybrid system. A yeast homolog will be expressed and analyzed to provide insight into the evolution of this family of neuronal calcium sensors.