Current Biology, Vol. 13, 1306–1310, August 5, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S 09 60 - 98 22 ( 03 )0 0 51 1- 6
Light-Mediated Activation of Rac-1 in Photoreceptor Outer Segments Nagaraj Balasubramanian and Vladlen Z. Slepak* Department of Molecular and Cellular Pharmacology and the Neuroscience Program University of Miami School of Medicine Miami, Florida 33136
Summary Small GTP binding proteins regulate diverse biological processes including gene expression, cytoskeleton reorganization, and protein and vesicular transport [1–4]. While small GTPases have been investigated in a wide variety of cells, few studies have addressed their role in photoreceptors. In vertebrate retinal rods, the light stimulus is transmitted from rhodopsin via the pathway mediated by the heterotrimeric G protein transducin [5–7]. To increase their sensitivity to light, photoreceptors accumulate remarkably high concentrations of rhodopsin and transducin in specialized cellular compartments, the outer segments (OS). Transport of these proteins from the inner segments is regulated by the small GTPases Rab6 and Rab8, which do not enter OS [8–10]. Here, we asked if small G proteins have other functions in photoreceptors. We show that OS contain the small GTPase Rac-1, a member of the Rho family. In contrast to other cells, Rac-1 in OS is exclusively associated with the membranes and resides in lipid rafts. Most importantly, Rac-1 is activated by light. This activation is specifically blocked by a synthetic peptide corresponding to the Asn-Pro-X-XTyr motif found in rhodopsin, and Rac-1 coprecipitates with rhodopsin on Concanavalin A Sepharose. These data provide the first direct evidence for the existence of a novel pathway activated by rhodopsin. Results and Discussion Presence of Rac-1 in Photoreceptor Outer Segments Western blot analysis of purified bovine outer segments (OS) revealed the presence of Rac-1 at a level similar to that seen in other cells, such as human embryonic kidney (HEK) and bovine aorta epithelial (BAE) cells (Figure 1A). The GDP dissociation inhibitor of Rac-1, RhoGDI, was not detected in OS. In OS, Rac-1 is exclusively found in the membranes in a pattern similar to a known membrane protein RGS9-1, whereas, in HEK293 cells, a significant fraction of Rac-1 is cytosolic (Figure 1B). It is unlikely that soluble Rac-1 was lost during the OS isolation procedure because phosducin, a soluble protein, was present in the same preparation. Indirect immunofluorescence microscopy of mouse retinal sections confirmed the localization of Rac-1 in OS (Figure 1C). As expected, Rac-1 is also present in the inner segments (IS) and the outer nuclear layer (ONL) along with another *Correspondence:
[email protected]
small G protein, Arf6, which is not detected in OS. These results show that Rac-1 is present in OS and prompt us to investigate the properties and potential function of Rac-1 in this highly specialized cellular compartment.
Activity and Membrane Association of Rac-1 in OS To determine if Rac-1 could be activated, we used an assay with the GST fusion of the Cdc42/Rac binding domain of the effector kinase PAK1 (GST-PBD), which can selectively capture the GTP bound form of Rac-1 on glutathione-agarose [11]. In this pull-down experiment, less than 10% of Rac-1 was active in untreated, darkadapted OS, but the addition of GTP␥S caused activation of up to 70% of the total Rac-1 (Figure 2A). Thus, Rac-1 in OS can be activated by GTP␥S. We next asked if the nucleotide binding status of Rac-1 in OS or its interactions with known protein binding partners could affect its exclusive membrane association. Neither GTP␥S nor GDPS altered the anchoring of Rac-1, whereas transducin, as expected, was eluted by GTP␥S (Figure S1 in the Supplemental Data available with this article online). Since it has been shown that Rac-1 remains cytosolic when bound to its GDP dissociation inhibitor RhoGDI [12], we reasoned that the restricted membrane localization of Rac-1 in OS could be explained by the apparent absence of RhoGDI (Figure 1A). We found that addition of recombinant RhoGDI to the OS membranes solubilized 30%–40% of Rac-1, and this effect was independent of Rac-1 GTP/GDP status. In a similar experiment, the addition of GST-PBD also led to the extraction of Rac-1, but only upon its activation by GTP␥S (Figure 2B). The ability of RhoGDI and GSTPBD to extract Rac-1 is interesting in light of the fact that Rac-1 is anchored to the OS membrane very tightly. As shown in Figure 2C, Rac-1 could not be eluted with 6 M urea or high pH, whereas RGS9-1, which is known for its tight membrane association, was eluted. Further characterization of Rac-1 binding to the membranes showed that it was primarily associated with the detergent-resistant membrane fraction (Figure 2D) representing lipid rafts, which are domains enriched in sphingolipids and cholesterol [13, 14]. Light affects the localization of some photoreceptor proteins, such as transducin, to the rafts [15, 16], but it did not affect that of Rac-1 (Figure 2D). In accord with Rac-1 partitioning to the lipid raft fraction, cholesterol depletion of OS with -methylcyclodextrin partially solubilized Rac-1 (Figure 2E). These findings are consistent with earlier studies in other cells suggesting that cholesterol and lipid rafts are involved in membrane targeting of Rac-1 [17, 18]. In addition, we found that regardless of the MCD concentration and the time of incubation, only 30%– 40% of Rac-1 can be extracted, and following cholesterol depletion, neither RhoGDI (Figure 2F) nor GST-PBD could further solubilize Rac-1. Reciprocally, preextraction of membranes with RhoGDI or GST-PBD rendered MCD ineffective, suggesting that RhoGDI, GST-PBD, and MCD act on the same pool of Rac-1, which consti-
Rac-1 in Photoreceptor Outer Segments 1307
Figure 1. Presence of Rac-1 in Photoreceptor Outer Segments (A) Western blot analysis of purified bovine photoreceptor OS, bovine aortal endothelial (BAE) cells, and human embryonic kidney (HEK) cells with antibodies against Rac-1, RhoGDI, and tubulin. (B) To obtain the cytosolic (C) and membrane (M) fractions, OS or HEK293 cells were sheared through an 18G needle and centrifuged. The supernatant and pellet fractions were analyzed by Western blot with antibodies to Rac-1, RGS9-1, and phosducin (Phd). The positions of prestained molecular markers (Bio Rad), in kilodaltons, are shown on the right. (C) Sections of retina from light-adapted mice. Panel 1, phase contrast; panels 2 and 3, indirect immunofluorescence with antibodies to ARF6 and Rac-1; panel 4, control staining without primary antibodies. Data are representative of three independent experiments.
tutes 30%–40% of the total Rac-1. This pool requires cholesterol and is sensitive to exogenously added RhoGDI or GST-PBD, indicating that there is another Rac-1 pool (60%–70%), which binds to the membranes independently of cholesterol and is insensitive to RhoGDI or GST-PBD. Rac-1 Coprecipitates with Rhodopsin and Is Activated by Light The small G proteins ARF6 and RhoA were shown to coimmunoprecipitate with some G protein-coupled receptors that have an Asn-Pro-X-X-Tyr amino acid motif in the seventh transmembrane domain [19]. Since this sequence is conserved in rhodopsin, we reasoned that Rac-1 could bind to rhodopsin, and upon immunoprecipitation of OS lysate with an anti-opsin antibody, Rac-1 was bound to the beads (data not shown). However, because the amount of the antibodies is limited, the beads can capture only a small fraction of total rhodopsin, which is present in OS at millimolar concentrations. Moreover, nonspecific binding of rhodopsin to Protein A beads with control antibodies was high, and Rac-1 bound as well, complicating the interpretation of the data. To circumvent these problems, we took advantage of the known ability of rhodopsin, a glycoprotein, to bind
to Concanavalin A Sepharose. The binding capacity of this resin is not limiting, and the nonspecific binding of rhodopsin to the beads in the presence of ␣-methylmannoside (AMM) was low (Figure 3A). In the Concanavalin A Sepharose pull-down assay, Rac-1 clearly cofractionated with rhodopsin (Figure 3B). Confirming the validity of this assay, a known rhodopsin-interacting protein, arrestin, specifically precipitated as well, whereas in a negative control, RGS9-1 only bound to the beads nonspecifically, regardless of the presence of AMM. Binding of Rac-1 was unaffected by preliminary extraction of membranes with GST-PBD (Figure 3C), RhoGDI, or MCD, indicating that a cholesterol-dependent pool of Rac-1 is distinct from the pool coprecipitating with rhodopsin. In the presence of GTP, illumination of dark-adapted OS caused activation of Rac-1, as shown by a dramatic increase of Rac-1 binding to GST-PBD in light (Figure 4A). Up to 50% of total Rac-1 present in the OS could be activated by light. A synthetic peptide (SAVYNPVIY IMMNKQ) encompassing the Asn-Pro-X-X-Tyr motif of rhodopsin blocked the light-mediated activation of Rac-1, whereas the solvent or an unrelated peptide had no effect (Figure 4B). The Asn-Pro-X-X-Tyr motif has recently been shown to participate in the light-induced conformational change in rhodopsin [20], and hence it
Current Biology 1308
Figure 2. Activation and Membrane Association of Rac-1 in OS (A) To test Rac-1 activation, OS were incubated without (⫺) or with (⫹) 1 mM GTP␥S and then solubilized with 80 mM N-octyl glucoside. The lysates were then mixed with purified GST-PBD (⫹) or buffer (⫺) and were added to Glutathione-Sepharose beads. The eluates from the beads were then probed for Rac-1 by using Western blot. (B) Solubilization of Rac-1 by RhoGDI and GST-PBD. Membranes were incubated with purified recombinant RhoGDI or GST-PBD (⫹) or without the proteins (⫺) in the presence (⫹) of 1 mM GDPS or GTP␥S or buffer (⫺) and were centrifuged, and the supernatants were probed for Rac-1. (C) OS membranes were treated with a control buffer, 0.1 M Na2CO3 (pH 11), or 6 M urea and were centrifuged. The supernatants and pellets were analyzed for the presence of Rac-1 and RGS9-1 by using Western blot. (D) Dark- or light-adapted OS were treated with 0.5% Triton X-100 and were fractionated on a sucrose density gradient to obtain detergent-resistant membranes (lipid rafts) as described in [16]. Fractions were probed with antibodies to Rac-1 and G␣t. (E) OS were incubated with (⫹) or without (⫺) 10 mM -methylcyclodextrin (MCD), centrifuged, and probed for the presence of Rac-1 and RGS9-1. (F) One aliquot of OS membranes was first extracted with (⫹) or without (⫺) RhoGDI and was centrifuged. The supernatant (first extract) was collected, and the pellet was extracted with 10 mM MCD and centrifuged to obtain the second extract and the pellet. The second aliquot of OS membranes was treated similarly, but it was first extracted by MCD, and then by RhoGDI. Collected fractions (first and second extracts, and the final pellets) were probed for the presence of Rac-1.
is still possible that rhodopsin activates Rac-1 indirectly. Indeed, the amount of Rac-1 that coprecipitated with rhodopsin on Concanavalin A Sepharose was not affected by either illumination or the Asn-Pro-X-X-Tyr peptide (Figure S3). While this does not rule out direct binding of Rac-1 to rhodopsin at a region distinct from the Asn-Pro-X-X-Tyr site or via another protein(s) in the complex, it does indicate that Rac-1 might associate with a different glycoprotein and be activated indirectly through a pathway downstream of rhodopsin. Mapping this novel pathway will require identification of Rac-1interacting proteins present in OS. Potential Function of Rac-1 in OS The key biological function of photoreceptor OS is generating a photoresponse, which occurs via the classic transducin-mediated pathway. What could be the potential role of Rac-1 in this system? Although a regulatory role in phototransduction cannot be ruled out at this point, we think that Rac-1 could be involved in a different process(es). Light sets in motion slow molecular events important for light adaptation, for example, active transport of proteins: transducin moves from the outer to inner segments, and arrestin travels in the oppo-
site direction [21–23]. Although, in general, protein traffic in photoreceptors is not well understood, it has been shown that rhodopsin movement to OS occurs via microtubules [24]. Since Rac-1 is known to regulate microtubules, actin cytoskeleton, and vesicular trafficking [3–5, 25, 26], it is a good candidate for being involved in various transport processes, such as protein trafficking and moving and/or shedding of the disks, which is a light-sensitive process [27]. In Drosophila, expression of constitutively active Rac-1 restores morphological changes resulting from a rhodopsin null mutation via an unidentified mechanism [28]. Our work brings out lightmediated activation of Rac-1 as a potential mechanism regulating cell morphology and perhaps other functions in normal vertebrate photoreceptors. Supplemental Data Supplemental Data describing the lack of the effect of GTP on membrane localization of Rac-1 and the Experimental Procedures used in this work are available at http://www.current-biology.com/cgi/ content/full/13/15/1306/DC1/. Acknowledgments We thank Drs. D. Espinosa, S. Cousins, and D. Nussenzveig for their help with imaging the mouse retinal sections, Drs. R. Molday, B.
Rac-1 in Photoreceptor Outer Segments 1309
Figure 3. Coprecipitation of Rac-1 with Rhodopsin on Concanavalin A Sepharose (A) OS were solubilized with 1% Triton X-100 and were incubated with Concavalin A (ConA) Sepharose in the presence (⫹) or absence (⫺) of 0.2 mM ␣-methylmannoside (AMM). No OS lysate was added to the beads to control for the protein bands originated from the immobilized ConA (⫺). The eluates from the beads were resolved by SDS-PAGE and were stained with Coomassie. The arrows indicate the bands of rhodopsin (Rhod) and Concanavalin A (Con A). (B) The eluates from the ConA beads incubated with (⫹) or without (⫺) AMM were tested for the presence of rhodopsin, Rac-1, arrestin, and RGS9-1. (C) Two aliquots of OS membranes were extracted with GST-PBD (⫹) or buffer (⫺) and centrifuged, and the pellets were solubilized with Triton (TX-100 extract), subjected to affinity chromatography on ConA Sepharose, and probed for Rac-1 as in (A).
Willardson, R. Khan, C. Smith, and P. Hargrave for providing antibodies, and Dr. S. Nair for helpful discussions. This work was supported by National Institutes of Health RO1 grants GM60019 and EY12982 (V.Z.S.) and by the American Heart Association, Florida Affiliate Postdoctoral Fellowship (N.B.). Received: March 18, 2003 Revised: May 19, 2003 Accepted: June 5, 2003 Published: August 5, 2003
Figure 4. Activation of Rac-1 by Light (A) OS were incubated in the dark or light and solubilized by 1% octylglucoside, and the aliquots were probed by Western for Rac-1 (“Total Rac-1”). The amount of active Rac-1 was then determined in the extracts by using the GST-PBD pull-down. Following the incubation and washes of the Glutahione-Sepharose, the amount of Rac-1 (“Active Rac-1”) bound to the beads was determined by Western blot. The histogram shows the relative band intensity (n ⫽ 4) of the active Rac-1 fraction in the dark- (closed bar) or lightexposed (open bar) OS as compared to total Rac-1 in the initial lysate. (B) The opsin-derived peptide SAVYNPVIYIMMNKQ (NPXXY) blocks the light-dependent activation of Rac-1. Activation of Rac-1 was assessed by using the GST-PBD method as in (A), but the OS were incubated in the presence of 0.1 mM opsin peptide (NPXXY), an unrelated peptide (control), or DMSO in a concentration equal to that in the peptide solutions (none).
9.
10.
References 1. Clerk, A., and Sugden, P.H. (2000). Small guanine nucleotidebinding proteins and myocardial hypertrophy. Circ. Res. 86, 1019–1023. 2. Reif, K., and Cantrell, D.A. (1998). Networking Rho family GTPases in lymphocytes. Immunity 8, 395–401. 3. Ridley, A.J. (2001). Rho GTPases and cell migration. J. Cell Sci. 114, 2713–2722. 4. Ridley, A.J. (2001). Rho family proteins: coordinating cell responses. Trends Cell Biol. 11, 471–477. 5. Baylor, D. (1996). How photons start vision. Proc. Natl. Acad. Sci. USA 93, 560–565. 6. Hurley, J.B. (2002). Shedding light on adaptation. J. Gen. Physiol. 119, 125–128. 7. Arshavsky, V.Y., Lamb, T.D., and Pugh, E.N., Jr. (2002). G proteins and phototransduction. Annu. Rev. Physiol. 64, 153–187. 8. Deretic, D., and Papermaster, D.S. (1993). Rab6 is associated
11.
12.
13.
14.
15.
with a compartment that transports rhodopsin from the transGolgi to the site of rod outer segment disk formation in frog retinal photoreceptors. J. Cell Sci. 106, 803–813. Deretic, D., Huber, L.A., Ransom, N., Mancini, M., Simons, K., and Papermaster, D.S. (1995). rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. J. Cell Sci. 108, 215–224. Moritz, O.L., Tam, B.M., Hurd, L.L., Peranen, J., Deretic, D., and Papermaster, D.S. (2001). Mutant rab8 impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods. Mol. Biol. Cell 12, 2341– 2351. Benard, V., and Bokoch, G.M. (2002). Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349–359. Del Pozo, M.A., Kiosses, W.B., Alderson, N.B., Meller, N., Hahn, K.M., and Schwartz, M.A. (2002). Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat. Cell Biol. 4, 232–239. Tsui-Pierchala, B.A., Encinas, M., Milbrandt, J., and Johnson, E.M., Jr. (2002). Lipid rafts in neuronal signaling and function. Trends Neurosci. 25, 412–417. Brown, D.A., and London, E. (2000). Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224. Seno, K., Kishimoto, M., Abe, M., Higuchi, Y., Mieda, M., Owada, Y., Yoshiyama, W., Liu, H., and Hayashi, F. (2001). Light- and guanosine 5⬘-3-O-(thio)triphosphate-sensitive localization of a
Current Biology 1310
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26. 27. 28.
G protein and its effector on detergent-resistant membrane rafts in rod photoreceptor outer segments. J. Biol. Chem. 276, 20813– 20816. Nair, K.S., Balasubramanian, N., and Slepak, V.Z. (2002). Signaldependent translocation of transducin, RGS9-1-G5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors. Curr. Biol. 12, 421–425. Grimmer, S., van Deurs, B., and Sandvig, K. (2002). Membrane ruffling and macropinocytosis in A431 cells require cholesterol. J. Cell Sci. 115, 2953–2962. Kumanogoh, H., Miyata, S., Sokawa, Y., and Maekawa, S. (2001). Biochemical and morphological analysis on the localization of Rac1 in neurons. Neurosci. Res. 39, 189–196. Mitchell, R., McCulloch, D., Lutz, E., Johnson, M., MacKenzie, C., Fennell, M., Fink, G., Zhou, W., and Sealfon, S.C. (1998). Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature 392, 411–414. Fritze, O., Filipek, S., Kuksa, V., Palczewski, K., Hofmann, K.P., and Ernst, O.P. (2003). Role of the conserved NPxxY(x)5,6F motif in the rhodopsin ground state and during activation. Proc. Natl. Acad. Sci. USA 100, 2290–2295. Sokolov, M., Lyubarsky, A.L., Strissel, K.J., Savchenko, A.B., Govardovskii, V.I., Pugh, E.N., Jr., and Arshavsky, V.Y. (2002). Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation. Neuron 34, 95–106. McGinnis, J.F., Matsumoto, B., Whelan, J.P., and Cao, W. (2002). Cytoskeleton participation in subcellular trafficking of signal transduction proteins in rod photoreceptor cells. J. Neurosci. Res. 67, 290–297. Mirshahi, M., Thillaye, B., Tarraf, M., de Kozak, Y., and Faure, J.P. (1994). Light-induced changes in S-antigen (arrestin) localization in retinal photoreceptors: differences between rods and cones and defective process in RCS rat retinal dystrophy. Eur. J. Cell Biol. 63, 61–67. Tai, A.W., Chuang, J.Z., Bode, C., Wolfrum, U., and Sung, C.H. (1999). Rhodopsin’s carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. Cell 97, 877–887. Daub, H., Gevaert, K., Vandekerckhove, J., Sobel, A., and Hall, A. (2001). Rac/Cdc42 and p65PAK regulate the microtubuledestabilizing protein stathmin through phosphorylation at serine 16. J. Biol. Chem. 276, 1677–1680. Gundersen, G.G. (2002). Microtubule capture: IQGAP and CLIP170 expand the repertoire. Curr. Biol. 12, R645–R647. Boesze-Battaglia, K., and Goldberg, A.F. (2002). Photoreceptor renewal: a role for peripherin/rds. Int. Rev. Cytol. 217, 183–225. Chang, H.Y., and Ready, D.F. (2000). Rescue of photoreceptor degeneration in rhodopsin-null Drosophila mutants by activated Rac1. Science 290, 1978–1980.
