Alexander Pulvermüller

Calcium-Dependent Assembly of Centrin-G-Protein Complex in Photoreceptor Cells

ABSTRACT Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges of the visual G-protein transducin between the outer and inner segment of rod photoreceptors occur through the narrow connecting cilium. Here we demonstrate that transducin colocalizes with the Ca2+-binding protein centrin 1 in a specific domain of this cilium. Coimmunoprecipitation, centrifugation, centrin overlay, size exclusion chromatography, and kinetic light-scattering experiments indicate that Ca2+-activated centrin 1 binds with high affinity and specificity to transducin. The assembly of centrin-G-protein complex is mediated by the βγ-complex. The Ca2+-dependent assembly of a G protein with centrin is a novel aspect of the supply of signaling proteins in sensory cells and a potential link between molecular translocations and signal transduction in general.

Binding of inositol phosphates to arrestin

Arrestin binds to phosphorylated rhodopsin in its light‐activated form (metarhodopsin II), blocking thereby its interaction with the G‐protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin‐rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5‐tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 μM and 5 μM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.

Helix Formation in Arrestin Accompanies Recognition of Photoactivated Rhodopsin

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed.

Centrins, A Novel Group Of Cat2,2+-Binding Proteins In Vertebrate Photoreceptor Cells

Changes in the intracellular Ca2+-concentration affect the visual signal transduction cascade directly or more often indirectly through Ca2+-binding proteins. Here we review recent findings on centrins in photoreceptor cells of the mammalian retina. Centrins are members of a highly conserved subgroup of the EF-hand superfamily of Ca2+-binding proteins commonly associated with centrosome-related structures. In vertebrate photoreceptor cells, centrins are also prominent components in the connecting cilium linking the light sensitive outer segment with the biosynthetically active inner segment compartment. Recent findings demonstrate that Ca2+-activated centrin forms a complex with the visual G-protein transducin in photoreceptor cells. This Ca2+-dependent assembly of G-proteins with centrin is a novel aspect of the supply of signaling proteins in sensory cells, and a potential link between molecular translocations and signal transduction in general.

Centrins, gatekeepers for the light-dependent translocation of transducin through the photoreceptor cell connecting cilium.

Centrins are members of a highly conserved subgroup of the EF-hand superfamily of Ca(2+)-binding proteins commonly associated with centrosome-related structures. In the retina, centrins are also prominent components of the photoreceptor cell ciliary apparatus. Centrin isoforms are differentially localized at the basal body and in the lumen of the connecting cilium. All molecular exchanges between the inner and outer segments occur through this narrow connecting cilium. Ca(2+)-activated centrin isoforms bind to the visual heterotrimeric G-protein transducin via an interaction with the betagamma-subunit. Ca(2+)-dependent assemblies of centrin/G-protein complexes may regulate the transducin movement through the connecting cilium. Formation of this complex represents a novel mechanism in regulation of translocation of signaling proteins in sensory cells, as well as a potential link between molecular trafficking and signal transduction in general.

Light-dependent CK2-mediated phosphorylation of centrins regulates complex formation with visual G-protein

Centrins are Ca2+-binding EF-hand proteins. All four known centrin isoforms are expressed in the ciliary apparatus of photoreceptor cells. Cen1p and Cen2p bind to the visual G-protein transducin in a strictly Ca2+-dependent way, which is thought to regulate light driven movements of transducin between photoreceptor cell compartments. These relatively slow motile processes represent a novel paradigm in light adaptation of photoreceptor cells. Here we validated specific phosphorylation as a novel regulator of centrins in photoreceptors. Centrins were differentially phosphorylated during photoreceptor dark adaptation. Inhibitor treatments revealed protein kinase CK2 as the major protein kinase mediating phosphorylation of Cen1p, Cen2p and Cen4p, but not Cen3p, at a specific target sequence. CK2 and ciliary centrins co-localize in the photoreceptor cilium. Direct binding of CK2 and centrins to ciliary microtubules may spatially integrate the enzyme-substrate specificity in the cilium. Kinetic light-scattering assays revealed decreased binding affinities of phosphorylated centrins to transducin. Furthermore, we show that this decrease is based on the reduction of Ca2+-binding affinities of centrins. Present data describe a novel regulatory mechanism of reciprocal regulation of stimulus dependent distribution of signaling molecules.

N‐terminal and C‐terminal Domains of Arrestin Both Contribute in Binding to Rhodopsin†

Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G‐protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time‐resolved light scattering. To accomplish site‐specific shielding of protein regions, in a first step all three wild‐type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the N‐ or the C‐terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.

Rhodopsin and Its Kinase

Publisher Summary RK is an important enzyme of photo transduction, and its role in human physiology is exquisitely characterized. In the study described in the chapter, many techniques were developed to isolate the enzyme, assay it, and characterize specific interactions with photolyzed Rho (Rho*). Several posttranslational modifications were characterized. With these basic techniques in hand, mechanistical, kinetic, and structural questions can be asked. For the signaling state of photoreceptors to return to the dark condition, Rho* and activated G proteins are turned off and cGMP is resynthesized. Phosphorylation of Rho* and binding of the capping protein arrestin (Arr) result in the termination of physiological responses. While Rho* must be turned off to prevent continued activation of the entire population of G protein, its phosphorylation and subsequent Arr quenching must be timed properly so that a given number of G protein molecules become activated by Rho*. All of these activation and quenching reactions occur within hundreds of milliseconds and are collectively termed “phototransduction.”

Insights into functional aspects of centrins from the structure of N-terminally extended mouse centrin 1

Centrins are members of the family of Ca(2+)-binding EF-hand proteins. In photoreceptor cells, centrin isoform 1 is specifically localized in the non-motile cilium. This connecting cilium links the light-sensitive outer segment with the biosynthetic active inner segment of the photoreceptor cell. All intracellular exchanges between these compartments have to occur through this cilium. Three-dimensional structures of centrins from diverse organisms are known, showing that the EF-hand motifs of the N-terminal domains adopt closed conformations, while the C-terminal EF-hand motifs have open conformations. The crystal structure of an N-terminally extended mouse centrin 1 (MmCen1-L) resembles the overall structure of troponin C in its two Ca(2+) bound form. Within the N-terminal extension in MmCen1-L, residues W24 and R25 bind to the C-terminal domain of centrin 1 in a target-protein-like geometry. Here, we discuss this binding mode in connection with putative interaction sites of the target-protein transducin and the self-assembly of centrins.

Crystallization and preliminary X-ray studies of mouse centrin1.

Centrins belong to a family of Ca2+-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure-function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW approximately 22 240 Da) could be crystallized at room temperature, using 20-25%(w/v) PEG 1500, 5-10%(v/v) ethylene glycol and 1-2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 A at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 A, beta = 109.4 degrees. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, VM, of 2.2 A3 Da(-1).