Nikolai O. Artemyev

A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene

Retinal cone photoreceptors mediate fine visual acuity, daylight vision, and color vision. Congenital hereditary conditions in which there is a lack of cone function in humans cause achromatopsia, an autosomal recessive trait, characterized by low vision, photophobia, and lack of color discrimination. Herein we report the identification of mutations in the PDE6C gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase as a cause of autosomal recessive achromatopsia. Moreover, we show that the spontaneous mouse mutant cpfl1 that features a lack of cone function and rapid degeneration of the cone photoreceptors represents a homologous mouse model for PDE6C associated achromatopsia.

Intrinsically disordered gamma-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure.

The retinal phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic αβ-subunits of PDE6 and the α-subunit of transducin (αt). Detailed structural analysis of PDEγ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEγ, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEγ46–87 when bound to αt in the transition-state complex. NMR mapping of the interaction between PDEγ46–87 and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74–87 of PDEγ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to αt, is left free at this stage. These results indicate that the interaction between PDEγ and αt during the phototransduction cascade involves the selection of preconfigured transient conformations.

Transducin Activation State Controls Its Light-dependent Translocation in Rod Photoreceptors

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-α subunit (Gtα)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtαQ200L. An illumination threshold for the Gtα movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtαQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtα, the GtαQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtαQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtα, and apparently, do not require Gtα interaction with RGS9 and PDE6.

The Trimeric GTP-binding Protein (Gq/G11) α Subunit Is Required for Insulin-stimulated GLUT4 Translocation in 3T3L1 Adipocytes

To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active Gq(Gq/Q209L), Gi (Gi/Q205L), and Gs (Gs/Q227L) α subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor Gi/Q205L and Gs/Q227L α subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of Gq/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory Gq/G11 α subunit-specific antibody but not a Gi or Gsα subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound Gq/G11, expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between Gq/G11 function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not Gq/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, Gq/Q209L did not induce thein vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP3. Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins Gq and/or G11.

Regulation of transducin GTPase activity by human retinal RGS.

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory γ-subunit of PDE (Pγ), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin α-subunit, Gtα GDPAlF 4 − , while its binding to GtαGTPγS or GtαGDP was weak. hRGSr and Pγ did not compete for the interaction with Gtα GDPAlF 4 − . Affinity of the Pγ-Gtα GDPAlF 4 −interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of Gtα GDPAlF 4 −binding to Pγ labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PγBC). Binding of hRGSr to Gtα GDPAlF 4 −complexed with PγBC resulted in a maximal ∼40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for Gtα GDPAlF 4 −(K d 35 nm). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s−1. Addition of Pγ to the reconstituted system reduced the GTPase level accelerated by hRGSr (k cat 0.085 s−1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 μm rhodopsin these rates exceeded 0.7 s−1. Our data suggest that effects of hRGSr on transducin’s GTPase activity are attenuated by Pγ but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.

Characterization of the G alpha(s) regulator cysteine string protein.

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Active sites of the cyclic GMP phosphodiesterase γ‐subunit of retinal rod outer segments

Monoclonal antibodies were prepared to the γ‐subunit of the cGMP phosphodiesterase. One of them γp‐1, suppresses the activation of phosphodiesterase through the α‐subunit of transducin. The γ‐subunit fragment 24–45 rich in Arg and Lys residues is involved in γp‐1 binding and is essential for the γ‐subunit interaction with transducin. Carboxypeptidase Y cleaves off seven amino acid residues from the C‐terminus of the γ‐subunit resulting in phosphodiesterase activation. Thus, the C‐terminal fragment of γ‐subunit participates in phosphodiesterase inhibition.

The Inhibitory γ Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pγ), which interact with the catalytic heterodimer (Pαβ) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pγ/Pαβ interactions play a critical role. The quaternary organization of the αβγγ heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pγ-binding regions on Pα and Pβ through photolabel transfer from various Pγ positions throughout the entire molecule. The Pγ N-terminal Val16–Phe30 region was found to interact with the Pαβ GAFa domain, whereas its C terminus (Phe73–Ile87) interacted with the Pαβ catalytic domain. The interactions of Pγ with these two domains were bridged by its central Ser40–Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pγ and Pαβ. Furthermore, a photocross-linked product αβγ(γ) was specifically generated by the double derivatized Pγ, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pγ molecule binds Pαβ in an extended linear interaction and may even interact with both Pα and Pβ simultaneously.

Characterization of the Gαs Regulator Cysteine String Protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Light-dependent compartmentalization of transducin in rod photoreceptors.

Three major visual signaling proteins, transducin, arrestin, and recoverin undergo bidirectional translocations between the outer segment and inner compartments of rod photoreceptors in a light-dependent manner. The light-dependent translocation of proteins is believed to contribute to adaptation and neuroprotection of photoreceptor cells. The potential physiological significance and mechanisms of light-controlled protein translocations are at the center of current discussion. In this paper, I outline the latest advances in understanding the mechanisms of bidirectional translocation of transducin and determinants of its steady-state distribution in dark- and light-adapted photoreceptor cells.