The Presence of a Leu-Gly-Asn Repeat–Enriched Protein (LGN), a Putative Binding Partner of Transducin, in ROD Photoreceptors

Abstract

purpose. Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat–enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells.

methods. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPγS binding.

results. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The α subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange.

conclusions. Photoreceptor inner segments contain LGN, which can bind to the α subunit of transducin and potentially regulate its function.

The rod phototransduction cascade is a classic G-protein–mediated pathway, where rhodopsin serves as a light-activated guanine nucleotide exchange factor (GEF) for the G protein transducin. Rhodopsin-activated transducin (Gt) stimulates the effector enzyme cGMP phosphodiesterase through sequestration of its inhibitory γ subunit (PDEγ), resulting in a breakdown of cGMP (for review, see Refs. ^{1 2} ). The decrease in cGMP levels closes cGMP-gated cation channels and causes a decline in intracellular Na⁺ and Ca²⁺ and hyperpolarization of the photoreceptor plasma membrane (reviewed in Ref. ³ ). 

Photoreceptor recovery is achieved by several mechanisms. The decreased concentration of cGMP is restored by retinal guanylate cyclase (for review, see Refs. ^{4 5 6} ). The active state of rhodopsin is terminated on rhodopsin phosphorylation by rhodopsin kinase (G protein coupled receptor kinase [GRK1]) and the subsequent binding of arrestin (for reviews, see Refs. ^{7 8} ). The guanosine triphosphate (GTP)-bound state of the G protein is terminated by GTP hydrolysis, which is accelerated by the photoreceptor-specific regulator of G protein signaling RGS9-1, which in turn acts as a guanosine triphosphatase (GTPase) activating protein (GAP) for transducin. ^{9 10 11}  

A new concept in photoreceptor light adaptation has emerged from studies of both vertebrate and invertebrate vision, where adaptation to different light levels occurs through the translocation of signaling proteins to and from the rod outer segment (reviewed in Ref. ¹² ). In dark-adapted animals, transducin is predominantly located in the rod outer segments, whereas illumination causes transducin to redistribute to the inner segment and nuclear layer. It is thought that the departure of transducin from the outer segment prevents photoresponse saturation by reducing the amount of available transducin. This translocation is reversed in dark conditions. ^{13 14 15 16}  

Recent studies have implicated a novel family of molecules, activator of G protein signaling (AGS) proteins, in the regulation of the G protein cycle (for review, see Refs. ^{17 18} ). Using a functional screen based on the pheromone-response pathway in Saccharomyces cerevisiae, investigators have identified three proteins that activate the pathway in the absence of a typical receptor. ¹⁹ AGS1 is a member of the superfamily of Ras proteins, which acts as a GEF for heterotrimeric G proteins. AGS2 is identical with the light chain component of the cytoplasmic motor protein dynein. The mechanism of regulation of G protein signaling by AGS2 is not understood. AGS3 binds to Gα subunits preferentially in their GDP-bound form, acts as guanine nucleotide dissociation inhibitor (GDI) for Gαi subunits, and competes for interaction with Gβγ thus facilitating the activation of Gβγ-dependent effectors. ^{20 21} AGS3 possesses a series of seven tetratrico peptide repeat (TPR) motifs and four 20 amino acid repeats termed G-protein regulator (GPR) motifs, also known as Go-Loco motifs. ^{20 21 22 23 24} Binding of AGS3 to Gαi occurs through the GPR domain, which is sufficient to stabilize the GDP-bound conformation of Gαi and act as a GDI. ^{21 22 25} Protein interaction studies and/or functional screens in yeast indicate that the AGS3 GPR motif interacts with Gαi1-3, but not Gαz, -12, -s, -q, or -16. ^{21 23} Biochemical analysis of the interaction of the recombinant GPR domain of AGS3 with Gαt in vitro has shown that AGS3 can inhibit the rates of rhodopsin-stimulated GTPγS binding to transducin through the reduced rate of dissociation of GDP from Gαt. ²⁶ Another GPR domain-containing protein was identified as a Gαi-binding protein in a yeast two-hybrid screen. Its deduced amino acid sequence contains 10 Leu-Gly-Asn repeats, and it has thus been named LGN. ²⁷ Similar to AGS3, LGN contains seven TPR domains in its N terminus followed by a linker region and four GPR motifs. The overall amino acid identity between AGS3 and LGN is 59%. While studies in Drosophila and Caenorhabditis elegans and in mammalian cells implicate LGN and some other GPR domain-containing proteins in mitotic spindle organization, very little is known about the role of LGN in regulation of G protein signaling in a physiological system. ^{28 29}  

The presence of AGS and related proteins in photoreceptor cells has not been investigated, and it is not known whether they play a role in regulation of Gt in vivo. Here, we show that photoreceptor inner segments contain LGN. We also provide evidence that LGN interacts with Gαt and can act as a GDI for transducin. 

Materials and Methods

Antibodies

Two antibodies against LGN were used: anti-LGN linker antibody was raised against a synthetic peptide corresponding to the linker region of LGN (Ser⁴²⁰-Lys⁴⁴⁹ SEILAKQKPLIAKPSAKLLFVNRLKGKKYK), ²¹ and and anti-LGN C terminus, was generated in rabbits against a C-terminal peptide (amino acids Phe⁶⁵³-His⁶⁷⁷ FGLKDFLQNNALLEFKNSGKKSADH) of human LGN. Both anti-LGN antibodies were affinity purified with their corresponding peptides. The generation of AGS3 and -1 polyclonal antibodies against synthetic peptides has been described previously. ²¹ The antibody against the α subunit of transducin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). 

Preparation of RIS-ROS Population

The RIS-ROS (the mixture of rod inner segments [RIS], and rod outer segments [ROS]) preparations were performed as described by Baker et al. ³⁰ Briefly, dark-adapted bovine retinas (Lawson Co., Lincoln, NE) were thawed in 50% sucrose in ROS buffer (10 mM HEPES [pH 7.2], 5 mM MgSO₄, and 25 mM KCl) supplemented with a protease inhibitor mixture (1 μg/μL pepstatin A, 1 μg/mL leupeptin, and 4 μg/mL aprotinin). The thawed retinas were vortexed for 1 minute to break off the ROS and then filtered through a cheesecloth. After centrifugation at 13,000g for 1 hour at 4°C, the crude ROS-RIS were collected. All the steps were performed under dim red light. 

Preparation of ROS Membranes

ROS were prepared from frozen bovine retinas, as described earlier, ^{31 32} and passed through a 21-gauge needle five times, and the suspension was centrifuged at 14,000 rpm in a table-top centrifuge for 15 minutes at 4°C. The pellet was further suspended in ROS buffer. The membranes were washed with 6 M urea to remove peripheral membrane proteins. 

Preparation of Cytosolic, Membrane, and Cytoskeletal Fractions

The RIS-ROS fraction was passed through a 21-gauge needle, and the suspension was centrifuged at 14,000 rpm for 10 minutes at 4°C. The collected supernatant was designated the cytosolic fraction. The pellet was solubilized in a lysis buffer containing 20 mm Tris (pH 7.4), 1 mM EDTA, 2 mM MgCl₂, and 1% Triton X-100, supplemented with protease inhibitors, and was centrifuged at 100,000g for 15 minutes. The supernatant contained the detergent-extracted membrane proteins. The detergent-insoluble pellet, which was highly enriched in tubulin, represented the cytoskeletal fraction. 

Purification of Recombinant GST-GPR

The glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli strain BL21 and purified on glutathione-Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ) using a standard protocol. GST-LGN-GPR was constructed by PCR using full-length cDNA for LGN as the template. Primers corresponding to the GPR domain of LGN (Ser⁴⁷⁶-His⁶⁷⁷; forward primer: 5′-GGGGAATTCAGTGCAGATACTATTGGAGATGAAGGG-3′; reverse primer: 5′-ATGCTCGAGCTAATGGTCTGCCGATTTTTTCCC-3′) were designed to add EcoRI and XhoI sites to the 5′ and 3′ ends, respectively, of LGN-GPR to fuse the LGN open reading frame with the open reading frame of GST in the pGEX4T1 vector. GST-LGN was constructed by PCR, with a full-length cDNA for LGN used as the template. Primers corresponding to full-length LGN (Arg²-His⁶⁷⁷; forward primer: 5′-AGAGAAGACCATTCTTTTCATGTTCGT-3′; reverse primer: 5′-CTAATGGTCTGCCGATTTTTTCCC-3′) were designed to add SalI and NotI sites to the 5′ and 3′ ends, respectively, and cloned downstream of GST into the pGEX4T3 vector. The expression of soluble full-length LGN was achieved by lowering the isopropyl-β-d-thiogalactopyranoside (IPTG) induction temperature to 22°C. The optimal IPTG concentration was 5 μM and the time of induction 10 hours. The purity of proteins was tested by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and found to be >95%. Protein concentrations were determined by a Bradford protein assay. 

GST Pull-Down Assay

Dark-adapted ROS membrane was incubated with GDP or GTPγS for 10 minutes on ice followed by illumination, and then lysed in buffer containing 20 mM Tris, 150 mM NaCl and 1% Triton X-100, supplemented with protease inhibitors. Triton X-100, supplemented with protease inhibitors. The lysate was preincubated with GST-GPR (1 μg) for 15 minutes on ice and followed by an incubation with glutathione-Sepharose (20 μL). The beads were washed three times with 1 mL lysis buffer, and the bound proteins were eluted with SDS-PAGE sample buffer. The eluates were analyzed by Western blot (1:1000 dilution of the primary anti-Gαt antibodies). 

Immunoprecipitation

The crude ROS lysates were precleared with protein A-Sepharose beads, and then incubated overnight, with constant mixing, with 1 μg of affinity-purified anti-LGN antibody (linker region) and 20 μL of protein A. The beads were washed twice with the lysis buffer and then eluted using 2× SDS-PAGE sample buffer. The input and eluates were resolved by 12% SDS-PAGE and analyzed by Western blot. 

Protein Fractionation with Linear Sucrose Gradient

A 100-μL aliquot of detergent-solubilized RIS-ROS was layered on top of a 5% to 20% linear sucrose gradient and centrifuged at 35,000 rpm for 16 hours in a rotor (model SW41; Beckman Coulter, Fullerton, CA). Serial fractions (0.5 mL) were collected starting from the top of the tube, resolved on SDS-PAGE, and analyzed by Western blot with anti-LGN (linker region). Sedimentation standards were run in parallel gradients and included thyroglobulin (19S) and catalase (11.5S) as standards. 

Immunofluorescence

Mouse eyes were fixed in 0.1 M cacodylate buffer (pH 7.2), containing 4% paraformaldehyde and 0.5% glutaraldehyde for 3 hours at room temperature, washed, and cryoprotected for 12 hours in 0.1 M cacodylate buffer (pH 7.2) containing 30% sucrose at 4°C. Eyecups were then embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetec, Torrance, CA) and sectioned at −18°C. Ten-micrometer sections were collected and incubated in blocking solution (PBS containing 1% BSA, 5% normal goat serum, and 0.3% Triton X-100) for 1 hour at room temperature. Sections were incubated with primary antibody diluted in PBS (1:100), with 1% BSA, 1% normal goat serum, and 0.1% Triton X-100 for either 2 hours at room temperature or overnight at 4°C. For LGN detection, the anti-LGN linker antibody ²⁸ was used and visualized with FITC-conjugated goat anti-rabbit IgG, and images were acquired on a laser scanning confocal microscope (LSM 510; Carl Zeiss Meditec, Oberkochen, Germany). 

GTPγS Binding Assay

Preparations of Gα_t and Gβγ subunits were obtained as described previously. ³¹ Urea-washed ROS membranes (10 nM rhodopsin) were incubated for 5 minutes at room temperature in 0.5 mL of 20 mM Tris-HCl (pH 7.6) buffer containing 150 mM NaCl and 10 mM MgSO₄, with or without the addition of varying concentrations of GST-GPR. GTPγS binding was initiated by the addition of [³⁵S]GTPγS (0.1 μCi/10 μL). Aliquots of 10 μL were withdrawn, and subjected to vacuum filtration, using 0.45-μm nitrocellulose filters (Millipore, Bedford, MA). The filters were then washed three times with 1 mL ice-cold buffer, dried, and counted in a liquid scintillation counter. Determination of GTPγS binding at each time point was performed in duplicate. 

Results

Our Western blot analysis of bovine and mouse retinal preparations showed the presence of two protein bands, 75 and 30 kDa, recognized by anti-LGN antibodies. At the same time, AGS1 and -3 proteins were not detected in the retina, whereas they were clearly present in the brain. Because AGS3 cDNA was previously amplified from a retinal cDNA library, ²⁶ these results suggest that, whereas AGS3 may also be present at lower amounts, it appears that the major protein representing this family of molecules in the retina is LGN. The 75-kDa protein identified by the anti-LGN antibodies corresponds to the full-length LGN containing the GPR and TPR motifs. ²⁸ The origin of the 30-kDa protein is not clear, but it possibly represents a splice variant of LGN that is similar to a previously described variant of AGS3 lacking the TPR domains ³³ (Fig. 1) . Both the 75- and 30-kDa species were detected by antibodies generated against two distinct parts of the LGN molecule, the linker region and the C terminus, confirming the presence of LGN in the retina. 

The presence of LGN in the retina was further investigated by immunofluorescence microscopy. LGN was mainly localized in the inner segments of mouse retina, outer nuclear layer, and synaptic terminals of photoreceptors, but was not detected in the outer segments. Light did not affect the distribution of LGN (Fig. 2) , whereas Gαt translocated from ROS to the inner segments in response to light, in agreement with previous reports. ^{13 14 15 16}  

We then studied the distribution of LGN between the cytosolic, membrane and cytoskeletal fractions in a crude rod cell preparation enriched in rod outer and inner segments (RIS-ROS fraction). The full-length LGN was present in all three fractions, whereas the 30-kDa variant was absent in the cytoskeletal fraction (Fig. 3A) . TPR domains, found in several other proteins, are known to mediate protein–protein interactions and assembly of multiprotein complexes. ³⁴ Because LGN has seven TPR motifs, we tested to determine whether LGN also exists as a part of a high-molecular-mass complex. The RIS-ROS lysate was analyzed by ultracentrifugation on a sucrose density gradient. We found that 10% to 15% of 75-kDa LGN migrated in the high-molecular-mass fractions in the range of 17S to 20S (Fig. 3B) . 

The presence of GPR motifs in LGN promotes its interaction with the Gαi family. Therefore, to investigate the potential interaction between LGN and transducin, we subjected ROS membrane lysates to pull-down assays, by using the GST fusions of full-length LGN and its GPR domain. In the presence of GDP, both GST-LGN and GST-GPR bound robustly to Gαt (Fig. 4A) , whereas Gαt did not bind to the beads with immobilized GST (not shown). When the lysate was prepared after incubation of membranes with GTPγS, no interaction was detected. This shows that LGN interacts exclusively with the GDP-bound form of Gαt. To investigate the interaction between endogenous LGN and transducin, we immunoprecipitated the Gα_t-LGN complex from the rod cell lysates, using either anti-Gαt (Fig. 4B)or anti-LGN (Fig. 4C)antibodies. LGN and Gαt co-immunoprecipitated in both versions of the assay, indicating that transducin and LGN interact in situ. Like its recombinant counterpart, endogenous LGN only bound to the GDP-bound transducin, as their co-immunoprecipitation was drastically reduced by GTPγS (Figs. 4B 4C) . 

To ascertain whether LGN can function as a GDI for transducin, we measured its effect on the rate of rhodopsin-induced [³⁵S]GTPγS binding to transducin. Figure 5shows that in the presence of the GST-GPR domain of LGN, GTPγS binding was significantly reduced, indicating that LGN can potentially act as a GDI for transducin. 

We next assessed the amount of LGN in photoreceptors relative to Gαt by quantitative Western blot analysis. There is no established technique for isolation of homogeneous intact photoreceptor cells, and because LGN is a ubiquitous protein, determining the exact amount of LGN in photoreceptors is difficult. Therefore, we estimated the expression level of LGN in our RIS-ROS preparation. We calibrated the Western blot signal with an anti-LGN antibody and known amounts of GST-LGN and used it to determine the amount of LGN in the RIS-ROS lysate (Fig. 6A) . In parallel, the amount of transducin in the same preparation was determined by Coomassie staining, using purified transducin as a standard (Fig. 6B) . Our results show that the ratio of LGN to transducin was approximately 1:15, an amount sufficient to exert an effect on transducin function. 

Discussion

We demonstrated the presence of LGN in photoreceptor cells and showed that LGN can interact with Gαt in its GDP-bound state. Our data also show that LGN can inhibit GTPγS binding to transducin, indicating that it can potentially act as a GDI for this G protein. Because it is not present in the outer segments, it is unlikely that LGN plays a direct role in the regulation of phototransduction. This is consistent with the intrinsic biochemical properties of Gαt, which cannot exchange GDP for GTP in the absence of active rhodopsin. In contrast, other members of the Gαi family can exchange GDP for GTP spontaneously, and therefore require a GDI to keep their basal activity low. 

Recent studies have demonstrated the translocation of Gt from the outer to inner segments in response to light. This process has been implicated in light adaptation, because it reduces the amount of transducin available to carry signals from rhodopsin to PDE. ¹⁶ Because LGN is not present in the outer segment, it can only interact with transducin on its arrival to the inner segment. The role of this interaction could be to prevent Gαt interference with other G-protein–mediated signaling events in the inner segment. Because Gαt translocation precedes that of transducin Gβγ, ¹⁶ it is possible that free Gαt competes with other Gα subunits for their Gβγ subunits. For efficient sequestration of Gαt, LGN must be present in amounts that are comparable to that of Gαt. We estimated the LGN-to-transducin ratio in our rod cell preparations to be approximately 1:15. However, this amount is likely to be a significant underestimation, because our RIS-ROS preparation was enriched in ROS and depleted of RIS. Furthermore, complete translocation of transducin to the inner segment requires extremely bright light. Under physiological conditions, only a small pool of Gαt moves to the inner segments, and so the relatively less-abundant LGN could still be sufficient for neutralizing the potential negative effects of free Gαt, at least until the arrival of transducin’s Gβγ. Moreover, because LGN has four GPR domains, it can potentially bind more than one Gαt. Indeed, AGS3 has been shown to bind four Gαi subunits simultaneously through its four GPR domains. ^{21 22} The reassociation of heterotrimeric transducin could occur on the arrival of Gβγ to the inner segment, eventually leading to the return of transducin to the outer segment. 

It is known that several TPR domain-containing proteins participate in formation of multimeric protein complexes, probably because of the interaction of TPR domains with common acceptor molecules. ³⁴ For example, interflagellar transport (IFT) in photoreceptors involves assembly of a large complex containing IFT88, a protein with 10 TPR motifs. Cycling of the IFT complex between the axoneme and cell body is thought to be associated with transport of essential “cargo” proteins. ³⁵ We hypothesized that LGN is involved in the transport of transducin, by serving as an adaptor protein linking the IFT complex with Gαt. However, LGN and IFT88 did not co- immunoprecipitate (data not shown), and so it appears that LGN is a part of a distinct large protein complex. Another interesting observation is that a significant portion of full-length LGN is associated with the cytoskeleton, whereas the 30-kDa species is not, probably because it lacks the TPR domains. 

While the presence of LGN in photoreceptor cells and its interaction with Gαt strongly suggests that it has a role in the regulation of transducin function, we cannot rule out that it regulates other G proteins or another cellular activity. Understanding the role of LGN in photoreceptors necessitates further experiments involving photoreceptor-specific knockout of the LGN gene and disruption of the transducin-LGN interaction using transgenic mice models. 

 Supported by NIH Grants EY012982 and GM 060019 (VZS), F32MH65092 (JBB) and NIH center Grant P30 EY014801. KSN is an American Heart Association Florida Affiliate Fellow 0225220B.

 Submitted for publication August 20, 2004; revised October 15, 2004; accepted October 21, 2004.

 Disclosure: K.S. Nair, None; A. Mendez, None; J.B. Blumer, None; D.H. Rosenzweig, None; V.Z. Slepak, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

The presence of LGN in bovine and mouse retina. Bovine brain (BB), bovine RIS-ROS (BR), and mouse RIS-ROS (MR) lysates were analyzed by Western blot with two different anti-LGN (anti-linker region and C terminus, CT), anti-AGS3 and anti-AGS1 antibodies. Positions of molecular weight standards, in kilodalton, are indicated to the left of the gel.

Figure 2.

 

Localization of LGN in the retina. Retinal sections were prepared from mice dark-adapted overnight or exposed to 30 minutes of constant illumination with white light (2000 lux) after dilation of the pupil. Top: the sections were stained with anti-LGN antibody, detected with the anti-rabbit secondary FITC-conjugated antibody. The sections were costained with anti-rhodopsin monoclonal primary (mAb 1D4) and Texas red–conjugated secondary antibodies. LGN (green); rhodopsin (red). Bottom: the sections were costained with anti-Gαt antibody detected with FITC-conjugated (green) and anti-rhodopsin antibody (red). Transducin signal was restricted to the rod outer segments in darkness where it completely colocalized with rhodopsin; on light exposure, transducin signal extended to the inner compartments. OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

Figure 3.

 

Distribution of LGN in subcellular fractions. (A) The rod cell preparation (RIS-ROS) was fractionated by centrifugation and Triton X-100 extraction to obtain the cytosolic (Cyt), membrane (Memb) and cytoskeletal (CSK) fractions. These fractions were analyzed by Western blot with anti-LGN linker antibody. (B) The retinal lysate was separated on a linear (5%–20%) sucrose gradient. Top: Coomassie staining of the fractions which shows the overall protein distribution. Positions of the standards separated on a parallel gradient, thyroglobulin (19S) and catalase (11.5S), are indicated by the arrows above the gel. Bottom: the same fractions were analyzed by Western blot with anti-LGN antibodies. The detergent-insoluble pellet representing cytoskeleton, was collected from the bottom of the tube and analyzed, along with other fractions, by Western blot. Representative of four similar experiments.

Figure 4.

 

Interaction between LGN and Gαt. (A) ROS membranes were preincubated with GDP or GTPγS, illuminated and lysed. The lysates were mixed with recombinant GST-LGN (full-length) or GST fusion of GPR domain of LGN and then incubated with glutathione-Sepharose. The beads were washed and eluted with SDS-PAGE sample buffer. Eluted proteins were analyzed by Western blot and probed with anti-GST antibody or the anti-Gαt antibody. (B) Co-immunoprecipitation of endogenous LGN-Gαt complex with anti-transducin antibody. The RIS-ROS lysates were obtained in the presence of GDP or GTPγS and immunoprecipitated with anti-Gαt antibody. The presence of LGN and Gαt in the input and eluted fractions was tested by Western blot with anti-LGN or anti-Gαt antibody. (C) LGN-Gαt complex was immunoprecipitated with anti-LGN (linker region) antibody from RIS-ROS lysates obtained as in (B). The presence of LGN and Gα_t in the fractions was tested with anti-LGN or anti-Gαt antibody.

Figure 5.

 

LGN-mediated inhibition of GTPγS binding to transducin. Urea-washed bovine ROS and transducin subunits were obtained. The membranes (10 nM rhodopsin) were mixed with purified Gαt (0.25 μM) and Gβγ (0.5 μM) in the absence (□) or the presence of 2.5 and 5 μM GST-GPR (▴ and •, respectively), followed by the addition of 5 μM [³⁵S]GTPγS. The aliquots were removed at the indicated times, and the amount of bound GTPγS was determined by vacuum filtration and scintillation counting.

Figure 6.

 

Relative amount of LGN in the retinal cell extract. (A) The indicated amounts of purified GST-LGN and the RIS-ROS lysate (15 and 30 μg total protein) were analyzed by Western blot with anti-LGN linker antibody, using chemiluminescence detection. The signal intensity was measured by film densitometry, and the amount of LGN present in crude ROS lysate was quantified based on the known amounts of GST-LGN. The data from two such experiments are summarized in the graph, where the data from the crude lysate analysis (□) are superimposed on the calibration curve obtained with GST-LGN (▴). (B) The amount of transducin present in the lysate was quantified similarly by measuring the intensity of Coomassie-stained protein bands corresponding to the known amounts of purified α subunit of transducin (□) and to Gα_t in the lysate (▴). For quantitative analysis of both LGN and Gαt, the test samples were resolved on the same gel as the calibration standards.

The authors thank Steve Lanier (Louisiana State University, New Orleans, LA) and Jeannie Chen (University of Southern California, Los Angeles, CA) for support and helpful discussions, and Joe Besharse (Medical College of Wisconsin, Milwaukee, WI) for the antibodies to the IFT88 protein.