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J Biol Chem, Vol. 275, Issue 10, 6969-6974, March 10, 2000

Loss of the Effector Function in a Transducin- Mutant Associated with Nougaret Night Blindness^*

Khakim G. Muradov and Nikolai O. Artemyev

From the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A missense mutation, G38D, was found in the rod transducin  subunit (G_t) in individuals with the Nougaret form of dominant stationary night blindness. To elucidate the mechanism of Nougaret night blindness, we have examined the key functional properties of the mutant transducin. Our data show that the G38D mutation does not alter the interaction between G_t and G_t or activation of transducin by photoexcited rhodopsin (R*). The mutant G_t has only a modestly (~2.5-fold) reduced k_cat value for GTP hydrolysis. The GTPase activity of G_tG38D can be accelerated by photoreceptor regulator of G protein signaling, RGS9. Analysis of the G_tG38D interaction with cGMP phosphodiesterase revealed marked impairment of the mutant effector function. G_tG38D completely fails to bind the inhibitory PDE  subunit and activate the enzyme. Altogether, our results demonstrate a novel molecular mechanism in dominant stationary night blindness. In contrast to known forms of the disease caused by constitutive activation of the visual cascade, the Nougaret form has its origin in attenuated visual signaling due to loss of effector function by transducin G38D mutant.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The visual transduction cascade in vertebrate rod photoreceptor cells is among the best understood G protein signaling systems. In the outer segments of rod photoreceptor cells (ROS),¹ the visual G protein, transducin (G_t), couples the light receptor, rhodopsin (R), to the effector enzyme, cGMP phosphodiesterase (PDE). Photolyzed rhodopsin (R*) binds G_t and induces GDP/GTP exchange on the G_t subunit, which upon dissociation from R* and G_t activates PDE. To activate the enzyme, G_t·GTP interacts with the inhibitory PDE  subunits (P) and prevents them from blocking catalytic sites on the PDE subunits. The following decrease in intracellular cGMP concentration leads to a closure of cGMP-gated channels in the ROS plasma membrane (1-3). An analogous visual cascade operates in cone photoreceptor cells. Unlike the cones that mediate high intensity daytime color vision, rod photoreceptors are designed for dim light vision during nighttime.

Defects in the genes encoding key components of the visual transduction cascade have been linked to a number of retinal diseases. Certain forms of retinitis pigmentosa, a progressive photoreceptor degeneration, are caused by mutations in rhodopsin (4), - and -subunits of PDE (5-7), or the -subunit of the cGMP-gated channel (8). Mutations in rhodopsin (9, 10), PDE (11), or G_t (12) have been identified in different forms of congenital stationary night blindness. Stationary night blindness is not associated with retinal degeneration and manifests itself in the inability to see in the dark, whereas daytime vision is largely unaffected. A missense G_t mutation, G38D, was found in the Nougaret form of congenital stationary night blindness (12). Initially, it was thought that Nougaret night blindness resulted from a loss of rod function. However, a recent study using electroretinographic analysis of Nougaret patients has indicated the presence of a detectable albeit subnormal rod function coupled with slight impairment of cone function (13). A clear outcome of the disease is a significant loss of rod light sensitivity (13).

Functional consequences of the G38D mutation for transducin signaling have not been examined, although a Gly residue at the corresponding position in other GTP-binding proteins has been actively investigated. This Gly is located within the conserved P-loop with the consensus sequence GXXXXK(S/T) that binds the - and -phosphate of GDP and GTP in the superfamily of G proteins (14). One of the most common transforming mutations in p21^ras is a substitution of the corresponding residue Gly-12 by Val. This mutation essentially blocks the GTPase activity of p21^ras and prevents its stimulation by GAPs leading to constitutive activation of p21^ras-mediated pathways (15-17). Recently, a similar observation has been made for the analogous mutant of G_i1, G42V, which hydrolyzes GTP with a 30-fold lower rate than that of the parent protein (18). The ability of G_i1G42V to inhibit the effector enzyme, adenylyl cyclase, was not evaluated. Interestingly, G_z, in which the Gly residue is replaced by Ser, has a characteristically low k_cat for GTP hydrolysis (19). The biochemical properties of p21^rasG12V and G_i1G42V seem to point to the constitutive activity of the G_tG38D mutant as a cause of Nougaret night blindness. However, the alternative possibility that the G_tG38D mutation leads to an inactive visual cascade remained. Such a possibility is supported by analysis of an analogous G_s mutant, G_sG49V. The rate of GTP hydrolysis for G_sG49V is ~4-fold lower than that for G_s, and the GTP-bound mutant is capable of activation of adenylyl cyclase in vitro (20). However, G_sG49V was a very poor activator of adenylyl cyclase when expressed in a cyc S49 cell line (21). The loss of light sensitivity of the Nougaret rod photoreceptors could potentially be caused either by the desensitization due to constitutive activity of G_t or by reduced ability to transduce a visual signal due to impaired G_t function.

To identify the molecular mechanism of Nougaret night blindness, we have introduced the G38D substitution into G_t-like, effector-competent, G_t/G_i chimeric -subunit. An extensive examination of the functional properties of this mutant has revealed a complete loss of the effector function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [³⁵S]GTPS (1160 Ci/mmol) and [³²P]NAD (1000 Ci/mmol) were purchased from Amersham Pharmacia Biotech. Restriction enzymes were from New England Biolabs. T4 DNA ligase was from Roche Molecular Biochemicals. Cloned Pfu DNA polymerase was from Stratagene. TPCK-treated trypsin was from Worthington. All other chemicals were from Sigma or Fisher. Bovine ROS membranes were prepared as described previously (22). Urea-washed ROS membranes (uROS) were prepared according to protocol in Yamanaka et al. (23). G_t was purified according to Kleuss et al. (24).

Site-directed Mutagenesis-- The G_t*G38D mutant was obtained using the pHis6-G_t* vector for expression of the Lys-248/Asp-251  His-244/Asn-247 (HN) mutant of G_t/G_i1 chimera 8 (25, 26) as a template in the first round of PCR amplification. A forward primer carrying a NcoI site and a start codon was paired with a reverse primer coding the G38D mutation. This PCR product was used as a forward primer in the second round of PCR with pHis6-G_t* as a template and a reverse primer containing a BamHI site overlapping codons for G_t-207-209. The PCR product was digested with NcoI and BamHI and subcloned into pHis6-G_t*. The mutant sequence was confirmed by automated DNA sequencing at the University of Iowa DNA Core Facility. For expression of G_t* and G_t*G38D, 500 ml of 2× TY medium was inoculated with 10 ml of overnight culture of BL21(DE3) cells transformed with pHis6-G_t* or pHis6-G_t*G38D. At A₆₀₀ = 0.8, expression was induced by 30 µM isopropyl-1-thio--D-galactopyranoside for 3 h at 24 °C. Cells were pelleted and kept frozen at 70 °C. Purifications of recombinant proteins were carried out as described previously (25, 26). Yields for 80-90% pure G proteins were normally 3-5 mg.

cDNA coding for RGS9-284-461 (27, 28) was PCR-amplified from the human retinal cDNA library (provided by J. Nathans, Johns Hopkins University, Baltimore, MD) using primers carrying the NdeI and BamHI restriction sites. The PCR product was subcloned into the pET15b vector for expression of RGS9-284-461 as a His-tagged protein in Escherichia coli. RGS9-284-461 was expressed and purified essentially as described previously (28).

In Vitro Transcription-Translation of G_t and G_tG38D-- In vitro transcription-translations were carried out in the TNT T7 quick-coupled reticulocyte lysate system (Promega) according to the manufacture's recommendations. A plasmid pHis6-G_tG38D for in vitro translation of G_tG38D was obtained by excising the BamHI/HindIII fragment from pHis6-G_t*G38D and replacing it with the corresponding fragment from pHis6-G_t (25) containing wild-type bovine G_t cDNA. Plasmids pHis6-G_t and pHis6-G_tG38D were added to the translation mixture at a concentration of 4 µg/ml. Transcription-translations were carried out for 2 h at 30 °C with addition of 20 µM methionine or, where indicated, 1 µM [³⁵S]methionine. Translation mixtures using cold methionine (450 µl) were combined with 50-µl aliquots of translation mixtures using [³⁵S]methionine and applied onto a MonoQ HR 5/5 column (Amersham Pharmacia Biotech) equilibrated with 30 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgSO₄. The ³⁵S-labeled G_t or G_tG38D were eluted with a 0-0.5 M NaCl/30-min gradient and concentrated using Microcon^® centrifugal filter devices with cutoff of 10,000 Da (Millipore). To determine levels of in vitro translation of G_t and G_tG38D, transcription-translations were carried out in the presence of 1 µM [³⁵S]methionine and varying concentrations (1-10 µM) of unlabeled methionine. The ³⁵S-labeled G_t or G_tG38D was then precipitated, and the percentage of incorporation of radioactive label was calculated by liquid scintillation counting according to the manufacture's (Promega) recommendations. Taking into account a ratio of 12 Met residues/G_t molecule, the calculated in vitro translation levels of G_t and G_tG38D were 25-30 nM.

Trypsin Protection Assay-- G_t* or G_t*G38D (20 µM) was incubated for 5 min at 25 °C in 20 mM HEPES buffer (pH 8.0) containing 130 mM NaCl, 50 µM GDP, and 5 mM MgSO₄ (buffer A). Where indicated, 10 mM NaF and 30 µM AlCl₃ were included in the buffer. The GTPS-bound G_t* or G_t*G38D were prepared by preincubation of G proteins (25 µM) with bleached uROS (1 µM rhodopsin) and G_t (3 µM) in the presence of 50 µM GTPS for 30 min at 25 °C, followed by centrifugation for 60 min at 100,000 × g to remove the membranes. For the trypsin-protection test of in vitro translated G_t or G_tG38D, 0.5-µl aliquots of translation mixtures with additions of 1 µM [³⁵S]methionine were diluted into 20 µl of buffer A. The GTPS-bound G_t or G_tG38D were prepared by preincubation of translation mixtures with bleached uROS (0.5 µM rhodopsin) and G_t (0.5 µM) in the presence of 10 µM GTPS for 10 min at 25 °C. Trypsin digestions were performed with 25 µg trypsin/ml for 15 min at 25 °C and stopped with simultaneous addition of SDS sample buffer and heat treatment (100 °C, 5 min).

GTPS Binding Assay-- G proteins (1 µM) alone, mixed with 2 µM G_t or 2 µM G_t and uROS membranes (100 nM rhodopsin) were incubated for 3 min at 25 °C. Binding reactions were started by addition of 5 µM [³⁵S]GTPS (0.2 Ci). Aliquots of 50 µl were withdrawn at the indicated times, mixed with 1 ml of ice-cold 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl and 10 mM MgSO₄, and passed through Whatman cellulose nitrate filters (0.45 µm). The filters were then washed two times with the same buffer (2 ml, ice-cold) and counted in a liquid scintillation counter after dissolution in a 3a70B mixture. The k_app values for the binding reactions were calculated by fitting the data to the equation, GTPS bound (% bound) = 100% (1  e^-kt).

Pertussis Toxin-catalyzed ADP-ribosylation-- G_t* or G_t*G38D (0.5 µM each) were mixed with G_t (0-3 µM) in 50 µl of 20 mM Tris-HCl (pH 8.0) buffer containing 2 mM MgSO₄, 2 mM dithiothreitol, 1 mM EDTA, 10 µM GDP, and 3 µg/ml pertussis toxin (preactivated with 100 mM dithiothreitol and 0.25% SDS for 10 min at 30 °C). The reaction was started by addition of 5 µM [³²P]NAD and allowed to proceed for 1 h at 25 °C. Afterward, reaction mixtures were diluted with 1 ml of ice-cold 20 mM Tris-HCl (pH 8.0) buffer containing 100 mM NaCl and filtered through Whatman cellulose-nitrate filters. The filters were washed four times with the same buffer and counted in a liquid scintillation counter. In some experiments, 10-µl aliquots were withdrawn from reaction mixtures, mixed with sample buffer for SDS-PAGE, and analyzed by electrophoresis in 12% gels.

Single-turnover GTPase Assay-- Single-turnover GTPase activity measurements were carried out in suspensions of uROS membranes (5 µM rhodopsin) reconstituted with G_t* or G_t*G38D (2 µM) and G_t (2 µM) essentially as described in Refs. 29 and 30. The GTPase reaction was initiated by addition of 50 nM [-³²P]GTP (0.2 µCi). The GTPase rate constants were calculated by fitting the experimental data to an exponential function: % GTP hydrolyzed = 100(1  e^-kt), where k is a rate constant for GTP hydrolysis.

Fluorescence Assays-- Fluorescence assays of interaction between G_t* or G_t*G38D and P labeled with 3-(bromoacetyl)-7-diethyl aminocoumarin at Cys68 (PBC) were performed on a F-2000 fluorescence spectrophotometer (Hitachi) in 1 ml of 20 mM HEPES (pH 7.5) buffer containing 100 mM NaCl, 5 mM dithiothreitol, and 4 mM MgCl₂ essentially as described in Ref. 31. The GTPS-bound G_t* or G_t*G38D were prepared as described for the trypsin-protection assay. When the AlF₄-activated G_t* or G_t*G38D were tested, the buffer contained 30 µM AlCl₃, 10 mM NaF, and 1 µM GDP. Fluorescence of PBC (10 nM) was monitored with excitation at 445 nm and emission at 495 nm. Concentration of PBC was determined using ₄₄₅ = 53,000.

PDE Activation Assay-- PDE was extracted from ROS membranes and purified as described previously (32). HoloPDE (0.2 nM) was reconstituted with G_t* or G_t*G38D (1-2 µM) and 2 µM G_t in suspensions of uROS membranes containing 2 µM rhodopsin. GTPS (10 µM) was added to the reaction mixture, and PDE activity was measured using 100 µM [³H]cGMP similarly as described previously (33).

Other Methods-- Protein concentrations were determined by the method of Bradford (34) using IgG as a standard or using calculated extinction coefficients at 280 nm. SDS-PAGE was performed by the method of Laemmli (35) in 12% acrylamide gels. Fitting of the experimental data was performed with nonlinear least squares criteria using GraphPad Prizm (version 2) software.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression and Trypsin Sensitivity of the G_t*G38D Mutant-- A mutation, G38D, was introduced into the G_t-like G_t/G_i1 chimeric protein (25), which contained ~94% of G_t residues including the two key G_t effector residues His-244 and Asn-247 (26). This chimeric G_t, referred to as G_t*, is fully functional and able to bind the P subunit and stimulate PDE activity comparably to native G_t (26). G_t* was chosen as a template for mutagenesis because it has efficient expression, which allows complete examination and comparison of functional properties of G_t* and G_t*G38D. Expression levels in E. coli of soluble mutant G_t*G38D were analogous to those of G_t* (~4-5 mg/liter of culture). Activation of G subunits induces a conformational change that protects the G switch II region from proteolysis with trypsin. Fig. 1 demonstrates the results of the trypsin-protection assay for G_t* and G_t*G38D. Both proteins were capable of undergoing an activational conformational change upon R*-induced binding of GTPS (Fig. 1). However, the GDP·AlF₄-induced conformation of G_t*G38D is different from that of G_t*, as indicated by the lack of its resistance to trypsin in the presence of AlF₄. Such a tryptic resistance pattern of G_t*G38D is consistent with similar results reported for the G42V mutant of G_i1 (18).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The trypsin-protection test for Gt* and the Gt*G38D mutant. Figure shows a SDS-polycrylamide gel (12%) stained with Coomassie Blue. Gt* and Gt*G38D (20 µM) were treated with trypsin (25 µg/ml) for 15 min at 25 °C in the presence of 50 µM GDP alone or 50 µM GDP, 30 µM AlCl3, and 10 mM NaF. The GTPS-bound Gt* and Gt*G38D were obtained as described under "Experimental Procedures."

Kinetics of R*-dependent GTPS binding to G_t* and G_t*G38D-- The ability of R* to activate G_t* and G_t*G38D was examined by measuring their GTPS-binding kinetics in the presence of G_t. The rate of GTPS-binding is controlled by a rate-limiting GDP release of G subunits. Similarly to native G_t (25), G_t* and G_t*G38D in the absence (data not shown) or in the presence of G_t (Fig. 2) displayed very slow intrinsic rates of GDP release as measured by GTPS binding. This observation, that the G38D mutation does not significantly affect a high affinity of G_t for GDP, is in agreement with the GDP binding properties of G_i1G42V (18). In the presence of uROS membranes (100 nM R*) and G_t (2 µM), the rates of GTPS binding were markedly enhanced (Fig. 2). The comparable R*-induced GTPS binding rates by G_t* (k_app = 0.17 min¹) and G_t*G38D (k_app = 0.11 min¹) indicate that rhodopsin recognition in the mutant G_t* is generally not impaired.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time courses of GTPS binding to Gt* and Gt*G38D. The binding of GTP S to Gt* (, ) and Gt*G38D (, ) (1 µM each) mixed with 2 µM Gt (, ) or 2 µM Gt and uROS membranes (100 nM rhodopsin) (, ) was initiated by addition of 5 µM [35S]GTPS. G-bound GTPS was counted by withdrawing aliquots at the indicated times and passing them through Whatman cellulose nitrate filters (0.45 µm). GTPS binding is expressed as percentage of maximal, calculated based on protein concentration. The calculated kapp values (min1) are: 0.0014 ± 0.0002 (), 0.17 ± 0.006 (), 0.0003 ± 0.0001 (), and 0.11 ± 0.008 ().

Pertussis Toxin-catalyzed ADP-ribosylation of G_t* and G_t*G38D-- Pertussus toxin effectively ADP-ribosylates G_t·GDP at Cys-347 (36, 37). The heterotrimeric complex, G_t is a notably better substrate than G_t alone (38), making ADP-ribosylation a useful tool to assess G_t interaction with G_t. The GTPS binding properties of G_t*G38D in the presence of R* and G_t indicated that the mutation did not grossly alter the G_t* binding to G_t. However, an excess of G_t was used in the binding assay to avoid the influence of potential defects in the G_t*G38D/G_t coupling on activation of G_t*G38D by R*. To further examine the interaction of G_t*G38D with G_t, we carried out a pertussis toxin-catalyzed ADP-ribosylation of G_t* and G_t*G38D in the presence of increasing concentrations of G_t. The dose dependences of G_t-supported ADP-ribosylation of G_t* and G_t*G38D were similar, suggesting that G_t*G38D retains intact interaction with G_t (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   The Gt-dependent ADP-ribosylation of Gt* and Gt*G38D. Pertussis toxin-catalyzed ADP-ribosylation of Gt* or Gt*G38D Gt* (0.5 µM) was carried out in the presence of increasing concentrations of Gt as described under "Experimental Procedures." A, [32P]ADP-ribosylation of Gt* and Gt*G38D is analyzed by liquid scintillation counting. B, aliquots from the ADP-ribosylation reaction mixtures were analyzed by SDS-PAGE in 12% gels followed by autoradiography.

GTPase Activity of G_t*G38D and Effects of RGS9-- Unaltered interaction of G_t*G38D with G_t and activation by R* allowed examination of mutant GTPase activity under single turnover conditions ([GTP] < [G_t*]). The GTPase activities of G_t* and G_t*G38D were measured in the reconstituted system with G_t and uROS membranes. uROS membranes lack the activity of a photoreceptor GAP, RGS9 (27). The calculated k_cat for GTP hydrolysis by G_t* was 0.020 ± 0.003 s¹ (Fig. 4A). G_t*G38D hydrolyzed GTP with a notably lower rate (k_cat of 0.008 ± 0.0004 s¹) (Fig. 4B). The reduction in the k_cat value for GTP hydrolysis caused by the G_t*G38D mutation (~2.5-fold) is proportional to that observed in the G49V mutant of G_s (20) but considerably smaller than a 30-fold decrease in the k_cat of the G_i1G42V mutant (18). The GTPase activity of the p21^rasG12V mutant is insensitive to the p21^ras GAP (17). We tested the effects of a truncated RGS9 protein (amino acids 284-461) containing the RGS domain on rates of GTP hydrolysis for G_t* and G_t*G38D. Addition of 5 µM RGS9-284-461 resulted in acceleration of the GTPase activity of G_t by 6-fold (k_cat 0.12 ± 0.01 s¹) (Fig. 4A) and of G_t*G38D by ~3-fold (k_cat 0.023 ± 0.002 s¹).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   GTP hydrolysis by Gt* and Gt*G38D and effects of RGS9-284-461. Single turnover GTPase activity measurements were carried out in suspensions of uROS membranes (5 µM rhodopsin) reconstituted with Gt* (A) or Gt*G38D (B) (2 µM each) and Gt (2 µM) in the absence () or in the presence of 5 µM RGS9-284-461 ().

Effector Properties of G_t*G38D-- A fluorescence read-out assay was used to study the interaction between G_t*G38D and the P subunit. It utilizes the P subunit labeled at Cys-68 with the fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (PBC) (31). Binding of G_t to PBC causes a large increase in the probe fluorescence. Using this assay, affinities of G_t*·GTPS (K_d 1.7 ± 0.3 nM) or G_t*·AlF₄ (K_d 3.2 ± 0.3) nM) for PBC were similar (Fig. 5). Remarkably, G_t*G38D in both the GTPS- and AlF₄-activated conformations showed no detectable interaction with PBC (Fig. 5). To test the possibility that G_t*G38D binds PBC without causing a fluorescence increase, we investigated the binding of G_t* to PBC in the presence of high concentrations of G_t*G38D. No competition between G_t* and G_t*G38D for binding to PBC was detected as the K_d value for the G_t*/PBC interaction was essentially unchanged in the presence of 100 nM G_t*G38D (Fig. 5A). This result suggests the G_t mutation leads to a loss of the effector function. To confirm this conclusion, we evaluated the ability of G_t*G38D to stimulate activity of holoPDE reconstituted with uROS membranes and G_t in the presence of GTPS. For comparison, G_t* (2 µM) was capable under these conditions of stimulating basal PDE activity by ~18-fold (Fig. 6). G_t*G38D failed to activate cGMP hydrolysis (Fig. 6). Moreover, excess of the GTPS-bound G_t*G38D did not interfere with activation of holoPDE by G_t*, further supporting the lack of competition between G_t* and G_t*G38D for the effector molecule (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Binding of Gt* and Gt*G38D to PBC. The relative fluorescence change (F/Fo) of PBC (10 nM) (excitation at 445 nm, emission at 495 nm) was determined after addition of increasing concentrations of, for A, Gt*·GTPS (), Gt*G38D·GTPS (), and Gt*·GTPS in the presence of 100 nM Gt*G38D·GTPS () or, for B, Gt*·GDP () and Gt*G38D·GDP () in the presence of AlF4.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effects of Gt* and Gt*G38D on PDE activity. The cGMP hydrolytic activity of rod holoPDE (0.2 nM) was measured in suspensions of uROS (2 µM rhodopsin) reconstituted with 2 µM Gt and 10 µM GTPS in the absence or presence of Gt* or Gt*G38D. The PDE activity is expressed as a percentage of that measured in the presence of Gt* (100%: 210 mol of cGMP/s mol of PDE).

Binding of PBC to in Vitro Translated G_t and G_tG38D-- We utilized in vitro translations of G_t and G_tG38D to confirm that the G38D mutation causes a loss of effector function not only in the background of the chimeric G_t* protein but in the wild-type G_t as well. In vitro translation is known to produce a functional G_t (39). Although yields of recombinant proteins using in vitro translation are typically very low, a high sensitivity fluorescence binding assay allows examination of the interaction between in vitro translated G_t and PBC. The calculated levels of in vitro translations for G_t and G_tG38D were 25-30 nM. The trypsin-protection assay for in vitro translated G_t and G_tG38D demonstrated patterns very similar to those of G_t* and G_t*G38D expressed in E. coli. Both G_t and G_tG38D displayed a characteristic trypsin-protection upon R*-induced binding of GTPS, but the GDP·AlF₄-bound conformation of G_tG38D was not resistant to trypsin (Fig. 7A). Addition of G_t, purified from an in vitro translation mixture, to PBC in the presence of AlF₄ led to a large fluorescence change, whereas an analogously prepared G_tG38D had no effect (Fig. 7B).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Properties of in vitro translated Gt and GtG38D. A, the trypsin-protection test. Translation mixtures containing Gt or GtG38D (0.5 µl each) were diluted into 20 µl of 20 mM HEPES buffer (pH 8.0) containing 130 mM NaCl and 5 mM MgSO4, and treated with trypsin (25 µg/ml) for 15 min at 25 °C in the presence of 10 µM GDP or 10 µM GDP with 30 µM AlCl3 and 10 mM NaF. The GTPS-bound Gt and GtG38D were obtained as described under "Experimental Procedures." The proteolytic patterns were analyzed using fluorography of 12% polyacrylamide gels. B, interaction of in vitro translated Gt and GtG38D with PBC. The relative fluorescence change (F/Fo) of PBC (10 nM) (excitation at 445 nm, emission at 495 nm) was determined after addition of Gt·GDP or GtG38D·GDP (8 nM each) in the presence of AlF4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heterotrimeric G proteins transduce a variety of extracellular signals (neurotransmitters, hormones, light, olfactory and taste signals) from specific cell surface receptors to intracellular effectors. Mutations in G subunits that lead to abnormal signaling are known to cause a large number of diseases (40). Abnormal G protein signaling might result from either attenuated or elevated signal transduction. Mutations of G subunits leading to lower protein stability, reduced ability for interaction with and activation by cognate receptors, inability to dissociate G, or diminished capacity to activate effectors would attenuate G protein transduction. Mutations causing an increase in spontaneous G GDP/GTP exchange, a lower GTPase activity, or impairment of interaction with GAP proteins are often a source of excessive G protein signaling.

Recently, a mutation in the visual G protein, G_tG38D, was found in patients with the Nougaret form of dominant stationary night blindness (12). Although psychophysical and electrophysiological consequences of the Nougaret night blindness have been thoroughly investigated (13), the molecular mechanism of the disease remained unclear. Despite a lack of biochemical characterization of G_tG38D, a wealth of information has been accumulated on mutations of an analogous Gly residue in small and heterotrimeric G proteins. The G12V mutation in p21^ras produces constitutively active signaling by inhibiting the p21^ras GTPase activity and abolishing its stimulation by GAP proteins (15-17). Likewise, the G_i1G42V mutant has a 30-fold lower k_cat for GTP hydrolysis in comparison to the wild-type G_i1 and it is insensitive to RGS proteins (18). By analogy with the p21^ras and G_i1 mutants, constitutive activity of G_tG38D becomes the most appealing model for abnormal function of rod photoreceptors in the Nougaret pedigree. Persistent activation of PDE by the GTP-bound G_tG38D would also provide a simple explanation to the dominant inheritance of the disease (12). Moreover, such a hypothesis would be consistent with existing biochemical evidence on dominant night blindness caused by two mutations in rhodopsin, G90D and A292E (9, 10). Both rhodopsin mutants are constitutively active and capable of activating transducin even in the absence of the retinal chromophore (9, 10). The PDE -subunit gene, a third gene implicated in dominant stationary night blindness, carries a missense mutation H258N (11). Although properties of the PDE mutant are not known, it has been suggested that it might be constitutively active due to impaired interaction with the P subunit (11). Finally, an Oguchi disease, a recessive form of stationary night blindness, was found to be associated with null mutations in the rhodopsin kinase gene (41). Rhodopsin kinase is intimately involved in inactivation of R*. Transgenic mice lacking rhodopsin kinase displayed larger and longer than normal single-photon responses (42). The excessive signaling is thought to saturate rod responses at abnormally low light-intensities in Oguchi disease (42). Similarly to light adaptation, constitutive activation of the visual cascade would cause desensitization of rod photoreceptors by lowering cGMP levels, keeping the cGMP-gated channels closed, the plasma membrane hyperpolarized, and lowering intracellular Ca²⁺ concentration. Supporting the hypothesis for constitutive activity of G_tG38D, some of the abnormalities seen in the rod and cone functions of Nougaret patients have been simulated by light adaptation of the normal retina (13). However attractive, constitutive activity toward effectors of G mutants with substitution of the Gly residue has not been firmly established. Examination of G_sG49V revealed that it can normally stimulate adenylyl cyclase in the reconstituted system (20), but only poorly in cyc S49 cells expressing the mutant (21).

To elucidate the biochemical mechanism of Nougaret night blindness, we investigated the key functional properties of G_t*G38D, such as interaction with R* and G_t, GTPase activity, interaction with RGS9, binding the P subunit, and the ability to stimulate PDE. G_t*G38D interaction with G_t and activation by R* was found largely intact. In comparison with G_t*, the mutant had only a very modest ~2.5-fold reduction in the k_cat value for GTP hydrolysis. The decrease in G_t*G38D GTPase activity was significantly smaller than that seen in the G_i1 G42V or G42S mutants (18). In addition, unlike G_i1G42V (18), G_t*G38D retained reduced ability to interact with RGS proteins, particularly, with a photoreceptor-specific RGS9. In contrast, the effector function of G_t*G38D is markedly impaired. G_t*G38D fails to bind P and activate PDE. The inability of the G38D mutant, in a background of wild-type G_t, to interact with the effector molecule was demonstrated using in vitro translated G_t and G_tG38D. Lack of trypsin protection of G_tG38D (or G_t*G38D) in the presence of AlF₄ indicates that the active conformation of the switch II region of the mutant G_t may differ from that in wild-type transducin. The switch II region of G_t is an essential effector binding domain (26, 43), and alterations in this region provide a plausible rationale for the loss of effector function. Moreover, similar patterns of sensitivity to trypsin between active conformations of G_i1G42V (18) and G_tG38D allow us to speculate that these mutations may have caused analogous conformational changes. A crystal structure of the GTPS bound G_i1G42V shows that the Val-42 side chain forces the peptide planes of the switch II residues 203-206 to rotate leading to disruption of ionic contacts between Arg-205 and the switch III/3-helix residues Asp-237 and Glu-245 (18). If a similar conformational change is caused by the G_tG38D mutation, it would break the linkage between the G_t switch II Arg-201 and the switch III/3-helix residues Glu-232 and Glu-241 (44). This linkage is central to the ability of G_t to assume the effector-competent conformation (26, 45). All mutations of the G_t switch II region residues that are involved in the linkage with switch III/3-helix have resulted in a severe impairment of G_t effector function (26).

An absolute inability of G_tG38D to activate PDE may only in part account for desensitization of rod photoreceptors in the Nougaret form of dominant night blindness. Heterozygous Nougaret patients, who presumably have about 50% functional transducin, display more than 99% reduction in rod sensitivity (13). The observed rod sensitivity loss implies that G_tG38D somehow interferes with expression of the wild-type allele, or the mutant has dominant negative properties. We demonstrated that G_t*G38D is unable to prevent PDE activation by G_t*. Despite the lack of evidence to support the dominant negative nature of the G38D mutant, such a possibility cannot be ruled out. G_t*G38D is fully capable of interaction with R*, and under dim light conditions and very low concentrations of R*, the mutant G_t may potentially compete with wild-type G_t for R*. Such a competition would slow the rate of formation of G_t·GTP and, consequently, attenuate PDE activation.

Overall, our results demonstrate a novel molecular mechanism in dominant stationary night blindness. In contrast to stationary night blindness caused by constitutive activation of the visual cascade by rhodopsin mutants (9, 10), the Nougaret form has its basis in decreased visual signaling due to loss of transducin effector function.

	ACKNOWLEDGEMENT

We thank Dr. M. Natochin for valuable discussion.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant EY-10843 and by National Institutes of Health Grant DK-25295 (to the Diabetes and Endocrinology Research Center of the University of Iowa).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 319-335-7864; Fax: 319-335-7330; E-mail: nikolai-artemyev@uiowa.edu.

	ABBREVIATIONS

The abbreviations used are: ROS, rod outer segment(s); G_t, rod G protein (transducin) -subunit; PDE, rod outer segment cGMP phosphodiesterase; P and P, and  subunits of PDE; R*, light-activated (bleached) rhodopsin; uROS, urea-stripped ROS membranes; PBC, P labeled with 3-(bromoacetyl)-7-diethyl aminocoumarin (BC); GTPS, guanosine 5'-O-(3-thiotriphosphate); RGS proteins, regulators of G protein signaling; GAP, GTPase-activating protein; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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