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1.
Small GTPases ; 12(1): 1-12, 2021 01.
Article in English | MEDLINE | ID: mdl-31068062

ABSTRACT

The small GTPase Arf4-based ciliary membrane-targeting complex recognizes specific targeting signals within sensory receptors and regulates their directed movement to primary cilia. Activated Arf4 directly binds the VxPx ciliary-targeting signal (CTS) of the light-sensing receptor rhodopsin. Recent findings revealed that at the trans-Golgi, marked by the small GTPase Rab6, activated Arf4 forms a functional complex with rhodopsin and the Arf guanine nucleotide exchange factor (GEF) GBF1, providing positive feedback that drives further Arf4 activation in ciliary trafficking. Arf4 function is conserved across diverse cell types; however, it appears that not all its aspects are conserved across species, as mouse Arf4 is a natural mutant in the conserved α3 helix, which is essential for its interaction with rhodopsin. Generally, activated Arf4 regulates the assembly of the targeting nexus containing the Arf GAP ASAP1 and the Rab11a-FIP3-Rabin8 dual effector complex, which controls the assembly of the highly conserved Rab11a-Rabin8-Rab8 ciliary-targeting module. It was recently found that this module interacts with the R-SNARE VAMP7, likely in its activated, c-Src-phosphorylated form. Rab11 and Rab8 bind VAMP7 regulatory longin domain (LD), whereas Rabin8 interacts with the SNARE domain, capturing VAMP7 for delivery to the ciliary base and subsequent pairing with the cognate SNAREs syntaxin 3 and SNAP-25. This review will focus on the implications of these novel findings that further illuminate the role of well-ordered Arf and Rab interaction networks in targeting of sensory receptors to primary cilia. Abbreviations: CTS: Ciliary-Targeting Signal; GAP: GTPase Activating Protein; GEF: Guanine Nucleotide Exchange Factor; RTC(s), Rhodopsin Transport Carrier(s); SNARE: Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor; TGN: Trans-Golgi Network.


Subject(s)
Cilia
2.
J Cell Sci ; 131(24)2018 12 10.
Article in English | MEDLINE | ID: mdl-30404838

ABSTRACT

The Arf4-rhodopsin complex (mediated by the VxPx motif in rhodopsin) initiates expansion of vertebrate rod photoreceptor cilia-derived light-sensing organelles through stepwise assembly of a conserved trafficking network. Here, we examine its role in the sorting of VAMP7 (also known as TI-VAMP) - an R-SNARE possessing a regulatory longin domain (LD) - into rhodopsin transport carriers (RTCs). During RTC formation and trafficking, VAMP7 colocalizes with the ciliary cargo rhodopsin and interacts with the Rab11-Rabin8-Rab8 trafficking module. Rab11 and Rab8 bind the VAMP7 LD, whereas Rabin8 (also known as RAB3IP) interacts with the SNARE domain. The Arf/Rab11 effector FIP3 (also known as RAB11FIP3) regulates VAMP7 access to Rab11. At the ciliary base, VAMP7 forms a complex with the cognate SNAREs syntaxin 3 and SNAP-25. When expressed in transgenic animals, a GFP-VAMP7ΔLD fusion protein and a Y45E phosphomimetic mutant colocalize with endogenous VAMP7. The GFP-VAMP7-R150E mutant displays considerable localization defects that imply an important role of the R-SNARE motif in intracellular trafficking, rather than cognate SNARE pairing. Our study defines the link between VAMP7 and the ciliary targeting nexus that is conserved across diverse cell types, and contributes to general understanding of how functional Arf and Rab networks assemble SNAREs in membrane trafficking.


Subject(s)
ADP-Ribosylation Factors/metabolism , Cilia/metabolism , Membrane Fusion/physiology , SNARE Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Organelles/metabolism , Protein Transport/physiology , R-SNARE Proteins/metabolism , Rhodopsin/metabolism
3.
J Cell Sci ; 130(23): 3975-3987, 2017 Dec 01.
Article in English | MEDLINE | ID: mdl-29025970

ABSTRACT

The small GTPase Arf4 and the Arf GTPase-activating protein (GAP) ASAP1 cooperatively sequester sensory receptor cargo into transport carriers targeted to primary cilia, but the input that drives Arf4 activation in this process remains unknown. Here, we show, by using frog retinas and recombinant human proteins, that during the carrier biogenesis from the photoreceptor Golgi/trans-Golgi network (TGN) a functional complex is formed between Arf4, the Arf guanine nucleotide exchange factor (GEF) GBF1 and the light-sensing receptor, rhodopsin. Rhodopsin and Arf4 bind the regulatory N-terminal dimerization and cyclophillin-binding (DCB)-homology upstream of Sec7 (HUS) domain of GBF1. The complex is sensitive to Golgicide A (GCA), a selective inhibitor of GBF1 that accordingly blocks rhodopsin delivery to the cilia, without disrupting the photoreceptor Golgi. The emergence of newly synthesized rhodopsin in the endomembrane system is essential for GBF1-Arf4 complex formation in vivo Notably, GBF1 interacts with the Arf GAP ASAP1 in a GCA-resistant manner. Our findings indicate that converging signals on GBF1 from the influx of cargo into the Golgi/TGN and the feedback from Arf4, combined with input from ASAP1, control Arf4 activation during sensory membrane trafficking to primary cilia.


Subject(s)
ADP-Ribosylation Factors/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Microtubule-Associated Proteins/metabolism , Rhodopsin/metabolism , Xenopus Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cilia/metabolism , Protein Transport/physiology , Retina/metabolism , Sensory Receptor Cells/metabolism , Xenopus , rab GTP-Binding Proteins/metabolism
4.
Small GTPases ; 6(4): 165-73, 2015 10 02.
Article in English | MEDLINE | ID: mdl-26399276

ABSTRACT

Small GTPases function as universal molecular switches due to the nucleotide dependent conformational changes of their switch regions that allow interacting proteins to discriminate between the active GTP-bound and the inactive GDP-bound states. Guanine nucleotide exchange factors (GEFs) recognize the inactive GDP-bound conformation whereas GTPase activating proteins (GAPs), and the GTPase effectors recognize the active GTP-bound state. Small GTPases are linked to each other through regulatory and effector proteins into functional networks that regulate intracellular membrane traffic through diverse mechanisms that include GEF and GAP cascades, GEF-effector interactions, common effectors and positive feedback loops linking interacting proteins. As more structural and functional information is becoming available, new types of interactions between regulatory proteins, and new mechanisms by which GTPases are networked to control membrane traffic are being revealed. This review will focus on the structure and function of the novel Rab11-FIP3-Rabin8 dual effector complex and its implications for the targeting of sensory receptors to primary cilia, dysfunction of which causes cilia defects underlying human diseases and disorders know as ciliopathies.


Subject(s)
Cell Membrane/metabolism , I-kappa B Kinase/metabolism , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , rab GTP-Binding Proteins/metabolism , Animals , Cell Membrane/genetics , Cilia/genetics , Cilia/metabolism , Ciliopathies/enzymology , Ciliopathies/genetics , Ciliopathies/pathology , Germinal Center Kinases , Humans , I-kappa B Kinase/genetics , Multienzyme Complexes/genetics , Protein Serine-Threonine Kinases/genetics , rab GTP-Binding Proteins/genetics
5.
J Cell Sci ; 128(7): 1375-85, 2015 Apr 01.
Article in English | MEDLINE | ID: mdl-25673879

ABSTRACT

Primary cilia have gained considerable importance in biology and disease now that their involvement in a wide range of human ciliopathies has been abundantly documented. However, detailed molecular mechanisms for specific targeting of sensory receptors to primary cilia are still unknown. Here, we show that the Arf and Rab11 effector FIP3 (also known as RAB11FIP3) promotes the activity of Rab11a and the Arf GTPase-activating protein (GAP) ASAP1 in the Arf4-dependent ciliary transport of the sensory receptor rhodopsin. During its passage out of the photoreceptor Golgi and trans-Golgi network (TGN), rhodopsin indirectly interacts with FIP3 through Rab11a and ASAP1. FIP3 competes with rhodopsin for binding to ASAP1 and displaces it from the ternary complex with Arf4-GTP and ASAP1. Resembling the phenotype resulting from lack of ASAP1, ablation of FIP3 abolishes ciliary targeting and causes rhodopsin mislocalization. FIP3 coordinates the interactions of ASAP1 and Rab11a with the Rab8 guanine nucleotide exchange factor Rabin8 (also known as RAB3IP). Our study implies that FIP3 functions as a crucial targeting regulator, which impinges on rhodopsin-ASAP1 interactions and shapes the binding pocket for Rabin8 within the ASAP1-Rab11a-FIP3 targeting complex, thus facilitating the orderly assembly and activation of the Rab11-Rabin8-Rab8 cascade during ciliary receptor trafficking.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Cilia/metabolism , Protein Serine-Threonine Kinases/metabolism , Rhodopsin/metabolism , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Carrier Proteins/genetics , Cilia/genetics , Dimerization , Germinal Center Kinases , Humans , Mice , Protein Binding , Protein Serine-Threonine Kinases/genetics , Rhodopsin/genetics , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , trans-Golgi Network/metabolism
6.
Prog Retin Eye Res ; 38: 1-19, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24135424

ABSTRACT

Rhodopsin is a key molecular constituent of photoreceptor cells, yet understanding of how it regulates photoreceptor membrane trafficking and biogenesis of light-sensing organelles, the rod outer segments (ROS) is only beginning to emerge. Recently identified sequence of well-orchestrated molecular interactions of rhodopsin with the functional networks of Arf and Rab GTPases at multiple stages of intracellular targeting fits well into the complex framework of the biogenesis and maintenance of primary cilia, of which the ROS is one example. This review will discuss the latest progress in dissecting the molecular complexes that coordinate rhodopsin incorporation into ciliary-targeted carriers with the recruitment and activation of membrane tethering complexes and regulators of fusion with the periciliary plasma membrane. In addition to revealing the fundamental principals of ciliary membrane renewal, recent advances also provide molecular insight into the ways by which disruptions of the exquisitely orchestrated interactions lead to cilia dysfunction and result in human retinal dystrophies and syndromic diseases that affect multiple organs, including the eyes.


Subject(s)
Macromolecular Substances/metabolism , Photoreceptor Connecting Cilium/physiology , Rhodopsin/metabolism , Rod Cell Outer Segment/physiology , Animals , Biological Transport/physiology , Humans , Protein Transport/physiology
7.
Small GTPases ; 4(2): 70-7, 2013.
Article in English | MEDLINE | ID: mdl-23567335

ABSTRACT

Small GTPases are versatile temporal and spatial regulators of virtually all cellular processes including signal transduction, cytoskeleton dynamics and membrane trafficking. They function as molecular switches, aided by a multitude of regulatory and effector proteins that link them into functional networks. A picture is beginning to emerge whereupon scaffold proteins with many functional domains perform the regulatory and effector functions, thus allowing the ordered recruitment and activation of small GTPases. This leads to the formation of scaffolding patches that coordinate cargo concentration and capture, with the recruitment and activation of the membrane tethering complexes and fusion regulators. This review will focus on the crosstalk of Arf and Rab GTPases at the Golgi complex and the scaffolds that facilitate their activation during trafficking of sensory receptors to primary cilia. The evolutionary conservation of the GTPase cascades in ciliogenesis and yeast budding will be discussed.


Subject(s)
ADP-Ribosylation Factors/metabolism , Cilia/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Golgi Apparatus/metabolism , Humans , Protein Transport
8.
EMBO J ; 31(20): 4057-71, 2012 Oct 17.
Article in English | MEDLINE | ID: mdl-22983554

ABSTRACT

Dysfunctional trafficking to primary cilia is a frequent cause of human diseases known as ciliopathies, yet molecular mechanisms for specific targeting of sensory receptors to cilia are largely unknown. Here, we show that the targeting of ciliary cargo, represented by rhodopsin, is mediated by a specialized system, the principal component of which is the Arf GAP ASAP1. Ablation of ASAP1 abolishes ciliary targeting and causes formation of actin-rich periciliary membrane projections that accumulate mislocalized rhodopsin. We find that ASAP1 serves as a scaffold that brings together the proteins necessary for transport to the cilia including the GTP-binding protein Arf4 and the two G proteins of the Rab family--Rab11 and Rab8--linked by the Rab8 guanine nucleotide exchange factor Rabin8. ASAP1 recognizes the FR ciliary targeting signal of rhodopsin. Rhodopsin FR-AA mutant, defective in ASAP1 binding, fails to interact with Rab8 and translocate across the periciliary diffusion barrier. Our study implies that other rhodopsin-like sensory receptors may interact with this conserved system and reach the cilia using the same platform.


Subject(s)
ADP-Ribosylation Factors/physiology , Adaptor Proteins, Signal Transducing/physiology , Cilia/physiology , Protein Serine-Threonine Kinases/physiology , Rod Cell Outer Segment/physiology , rab GTP-Binding Proteins/physiology , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Animals , Cattle , Cells, Cultured , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Germinal Center Kinases , Golgi Apparatus/metabolism , Kidney Tubules, Collecting/cytology , Mice , Mice, Knockout , Models, Molecular , Photoreceptor Connecting Cilium/physiology , Protein Binding , Protein Interaction Mapping , Protein Transport , RNA Interference , RNA, Small Interfering/pharmacology , Ranidae , Recombinant Fusion Proteins/metabolism , Retinal Photoreceptor Cell Outer Segment , Rhodopsin/chemistry , Rhodopsin/genetics , Rhodopsin/metabolism , Rod Cell Outer Segment/ultrastructure
9.
Vision Res ; 75: 5-10, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-22892112

ABSTRACT

This review will focus on the conserved molecular mechanisms for the specific targeting of rhodopsin and rhodopsin-like sensory receptors to the primary cilia. We will discuss the molecular assemblies that control the movement of rhodopsin from the central sorting station of the cell, the trans-Golgi network (TGN), into membrane-enclosed rhodopsin transport carriers (RTCs), and their delivery to the primary cilia and the cilia-derived sensory organelle, the rod outer segment (ROS). Recent studies reveal that these processes are initiated by the synergistic interaction of rhodopsin with the active form of the G-protein Arf4 and the Arf GTPase activating protein (GAP) ASAP1. During rhodopsin progression, ASAP1 serves as an activation platform that brings together the proteins necessary for transport to the cilia, including the Rab11a-Rabin8-Rab8 complex involved in ciliogenesis. These specialized molecular assemblies, through successive action of discrete modules, cooperatively determine how rhodopsin and other rhodopsin-like signaling receptors gain access to primary cilia.


Subject(s)
Photoreceptor Connecting Cilium/physiology , Retinal Photoreceptor Cell Outer Segment/physiology , Rhodopsin/metabolism , ADP-Ribosylation Factors/metabolism , Animals , Biological Transport/physiology , GTPase-Activating Proteins/metabolism , Humans , Protein Transport/physiology , rab GTP-Binding Proteins/metabolism , trans-Golgi Network/metabolism
10.
J Biomed Opt ; 17(6): 060504, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22734727

ABSTRACT

This study is to test anatomic correlates, including connecting cilium (CC) and inner segment (IS) ellipsoid, to the hyper-reflective band visualized by optical coherence tomography (OCT) and commonly attributed to the photoreceptor inner/outer segment (IS/OS) junction. A line-scan OCT (LS-OCT) was constructed to achieve sub-cellular resolution (lateral: ≈ 2 µm; axial: ≈ 4 µm) of excised living frog retinas. An electro-optic phase modulator was employed for rapid and vibration-free phase modulation. Comparison of normalized distance measurements between LS-OCT images and histological images revealed that the dominant source of the signal reported as the IS/OS OCT band actually originates from the IS.


Subject(s)
Retina/physiology , Retinal Photoreceptor Cell Outer Segment/physiology , Tomography, Optical Coherence/methods , Algorithms , Animals , Electrophysiology/methods , Equipment Design , Light , Optics and Photonics , Photoreceptor Cells, Vertebrate/pathology , Ranidae , Reproducibility of Results , Retina/anatomy & histology , Signal Processing, Computer-Assisted
11.
Mol Biol Cell ; 22(18): 3289-305, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21775626

ABSTRACT

Primary cilia regulate epithelial differentiation and organ function. Failure of mutant polycystins to localize to cilia abolishes flow-stimulated calcium signaling and causes autosomal dominant polycystic kidney disease. We identify a conserved amino acid sequence, KVHPSST, in the C-terminus of polycystin-1 (PC1) that serves as a ciliary-targeting signal. PC1 binds a multimeric protein complex consisting of several GTPases (Arf4, Rab6, Rab11) and the GTPase-activating protein (GAP), ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) in the Golgi, which facilitates vesicle budding and Golgi exocytosis. A related N-terminal ciliary-targeting sequence in polycystin-2 similarly binds Arf4. Deletion of the extreme C-terminus of PC1 ablates Arf4 and ASAP1 binding and prevents ciliary localization of an integral membrane CD16.7-PC1 chimera. Interactions are confirmed for chimeric and endogenous proteins through quantitated in vitro and cell-based approaches. PC1 also complexes with Rab8; knockdown of trafficking regulators Arf4 or Rab8 functionally blocks CD16.7-PC1 trafficking to cilia. Mutations in rhodopsin disrupt a similar signal and cause retinitis pigmentosa, while Bardet-Biedl syndrome, primary open-angle glaucoma, and tumor cell invasiveness are linked to dysregulation of ASAP1 or Rab8 or its effectors. In this paper, we provide evidence for a conserved GTPase-dependent ciliary-trafficking mechanism that is shared between epithelia and neurons, and is essential in ciliary-trafficking and cell homeostasis.


Subject(s)
Cilia/metabolism , Conserved Sequence , Multiprotein Complexes/metabolism , Protein Sorting Signals , Protein Transport , TRPP Cation Channels/metabolism , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Cell Line , Dogs , GTPase-Activating Proteins/metabolism , Gene Expression , Microtubule-Associated Proteins/metabolism , Molecular Sequence Data , Primary Cell Culture , Protein Binding , Protein Interaction Domains and Motifs , RNA Interference , Recombinant Fusion Proteins/metabolism , TRPP Cation Channels/chemistry , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism
12.
J Cell Sci ; 122(Pt 12): 2003-13, 2009 Jun 15.
Article in English | MEDLINE | ID: mdl-19454479

ABSTRACT

The biogenesis of cilia-derived sensory organelles, the photoreceptor rod outer segments (ROS), is mediated by rhodopsin transport carriers (RTCs). The small GTPase Rab8 regulates ciliary targeting of RTCs, but their specific fusion sites have not been characterized. Here, we report that the Sec6/8 complex, or exocyst, is a candidate effector for Rab8. We also show that the Qa-SNARE syntaxin 3 is present in the rod inner segment (RIS) plasma membrane at the base of the cilium and displays a microtubule-dependent concentration gradient, whereas the Qbc-SNARE SNAP-25 is uniformly distributed in the RIS plasma membrane and the synapse. Treatment with omega-3 docosahexaenoic acid [DHA, 22:6(n-3)] causes increased co-immunoprecipitation and colocalization of SNAP-25 and syntaxin 3 at the base of the cilium, which results in the increased delivery of membrane to the ROS. This is particularly evident in propranolol-treated retinas, in which the DHA-mediated increase in SNARE pairing overcomes the tethering block, including dissociation of Sec8 into the cytosol. Together, our data indicate that the Sec6/8 complex, syntaxin 3 and SNAP-25 regulate rhodopsin delivery, probably by mediating docking and fusion of RTCs. We show further that DHA, an essential polyunsaturated fatty acid of the ROS, increases pairing of syntaxin 3 and SNAP-25 to regulate expansion of the ciliary membrane and ROS biogenesis.


Subject(s)
Docosahexaenoic Acids/pharmacology , Fatty Acids, Omega-3/pharmacology , Qa-SNARE Proteins/metabolism , Rhodopsin/metabolism , Rod Cell Outer Segment/physiology , Synaptosomal-Associated Protein 25/metabolism , Animals , Cilia/metabolism , Cilia/physiology , Models, Biological , Organelles/metabolism , Organelles/physiology , Protein Binding/drug effects , Protein Binding/physiology , Protein Transport , Qa-SNARE Proteins/physiology , Ranidae , Rod Cell Outer Segment/metabolism , Synaptosomal-Associated Protein 25/physiology
13.
EMBO J ; 28(3): 183-92, 2009 Feb 04.
Article in English | MEDLINE | ID: mdl-19153612

ABSTRACT

Dysfunctions of primary cilia and cilia-derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans-Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase-activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post-TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1-mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4-based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.


Subject(s)
ADP-Ribosylation Factors/metabolism , Cilia/metabolism , Rhodopsin/chemistry , Rhodopsin/metabolism , Xenopus Proteins/metabolism , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cilia/ultrastructure , GTPase-Activating Proteins/metabolism , Guanosine Triphosphate/metabolism , Hydrolysis , I-kappa B Kinase/chemistry , I-kappa B Kinase/metabolism , Intracellular Membranes/metabolism , Intracellular Membranes/ultrastructure , Molecular Sequence Data , Mutant Proteins/metabolism , Organ Specificity , Protein Binding , Protein Sorting Signals , Protein Structure, Tertiary , Protein Transport , Retinal Degeneration/metabolism , Xenopus/genetics , rab GTP-Binding Proteins/metabolism , trans-Golgi Network/ultrastructure
14.
Methods Cell Biol ; 94: 241-57, 2009.
Article in English | MEDLINE | ID: mdl-20362094

ABSTRACT

Primary cilia and cilia-derived sensory organelles are cell's antennas that contain sensory receptors and signal transduction modules. Defects in the expression and targeting of ciliary proteins to this specialized cellular compartment lead to human disorders collectively known as ciliopathies. To examine the molecular basis for the ciliary targeting of the light receptor rhodopsin, we have developed a cell-free assay that reconstitutes its packaging into the specific post-Golgi rhodopsin transport carriers (RTCs). This assay accurately reproduces the in vivo process of carrier budding, while allowing examination of individual components of the macromolecular complexes, thus providing insight into a more general mechanism for the regulation of ciliary membrane targeting. Examples are shown for the use of this assay in rhodopsin trafficking. The cell-free assay is applicable to other ciliary-targeted sensory molecules.


Subject(s)
Biological Assay , Cell Fractionation , Cilia/metabolism , Rhodopsin/metabolism , Animals , Biological Assay/instrumentation , Biological Assay/methods , Cell Fractionation/instrumentation , Cell Fractionation/methods , Humans , Protein Transport/physiology , Ranidae , Retinal Rod Photoreceptor Cells/cytology , Rod Cell Outer Segment/chemistry , Rod Cell Outer Segment/ultrastructure , Subcellular Fractions/chemistry , trans-Golgi Network/metabolism
15.
Vision Res ; 46(27): 4427-33, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17010408

ABSTRACT

This review summarizes the most recent progress in the understanding of the role of rhodopsin C-terminal domain in the regulation of intracellular trafficking and photoreceptor morphogenesis. A proposed cascade of molecular interactions, initiated by the rhodopsin C-terminal sequence VXPX-COOH during trafficking from the Golgi/TGN in retinal photoreceptors, is relayed by the small GTPase ARF4 to the downstream effectors. One of the candidates for an ARF4 effector is the ARF-GAP ASAP1, which may function as a subunit of, or form a novel protein coat involved in trafficking from the TGN and in cytoskeletal remodeling, whose assembly is regulated by the binding of ARF4 to rhodopsin, and whose function is essential for the polarized trafficking toward the ROS.


Subject(s)
Rhodopsin/physiology , Rod Cell Outer Segment/metabolism , Animals , Cell Membrane/metabolism , Cell Polarity/physiology , Humans , Mutation , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Protein Structure, Tertiary/genetics , Protein Transport , Rhodopsin/genetics , Vision, Ocular/physiology
16.
Proc Natl Acad Sci U S A ; 102(9): 3301-6, 2005 Mar 01.
Article in English | MEDLINE | ID: mdl-15728366

ABSTRACT

The maintenance of photoreceptor cell polarity is compromised by the rhodopsin mutations causing the human disease autosomal dominant retinitis pigmentosa. The severe form mutations occur in the C-terminal sorting signal of rhodopsin, VXPX-COOH. Here, we report that this sorting motif binds specifically to the small GTPase ARF4, a member of the ARF family of membrane budding and protein sorting regulators. The effects of blocking ARF4 action were functionally equivalent to the effects of blocking the rhodopsin C-terminal sorting signal. ARF4 was essential for the generation of post-Golgi carriers targeted to the rod outer segments of retinal photoreceptors. Thus, the severe retinitis pigmentosa alleles that affect the rhodopsin sorting signal interfere with interactions between ARF4 and rhodopsin, leading to aberrant trafficking and initiation of retinal degeneration.


Subject(s)
ADP-Ribosylation Factors/metabolism , Mutation , Rhodopsin/physiology , Amino Acid Sequence , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Golgi Apparatus/metabolism , Microscopy, Confocal , Protein Binding , Protein Transport , Ranidae , Reactive Oxygen Species , Rhodopsin/chemistry , Rhodopsin/genetics , Rhodopsin/metabolism
17.
Mol Biol Cell ; 15(1): 359-70, 2004 Jan.
Article in English | MEDLINE | ID: mdl-13679519

ABSTRACT

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.


Subject(s)
Microfilament Proteins/metabolism , Phosphatidylinositols/metabolism , Phosphoproteins/metabolism , Rhodopsin/metabolism , rac1 GTP-Binding Protein/metabolism , Actins/metabolism , Animals , Cytoskeletal Proteins , Cytosol/metabolism , Enzyme Activation/drug effects , Lysophospholipids/pharmacology , Mass Spectrometry , Membrane Fusion/physiology , Microscopy, Confocal , Microscopy, Electron , Mutation , Phosphatidate Phosphatase/drug effects , Phosphatidate Phosphatase/metabolism , Phospholipase D/drug effects , Phospholipase D/metabolism , Propranolol/pharmacology , Protein Transport/physiology , Rod Cell Outer Segment , Sphingosine/analogs & derivatives , Sphingosine/pharmacology , rab GTP-Binding Proteins
18.
Invest Ophthalmol Vis Sci ; 43(5): 1655-61, 2002 May.
Article in English | MEDLINE | ID: mdl-11980887

ABSTRACT

PURPOSE: To test whether high levels of cAMP promote apoptosis and shorten the life of retinal rod photoreceptors, the changes in cAMP levels during retinal degeneration were analyzed in two transgenic rat models that express rhodopsin P23H and S334ter mutations. METHODS: Dark- and light-adapted heterozygous P23H (lines 1 and 3; P23H-1 and -3), S334ter line 4 (S334ter-4), and Sprague-Dawley (control) rats were studied at 4 to 8 weeks by cAMP enzyme competitive immunoassay and by cAMP immunocytochemistry. RESULTS: In control animals retinal cAMP content reached a steady state level at 30 days of age. Dark-adapted control retinas had up to 97% higher cAMP content than light-adapted retinas, and photoreceptor cells were the major source of this increase. Dark-adapted photoreceptors in all three lines of transgenic rats at advanced stages of retinal degeneration had cAMP content different from that of the control. In rats that express mutant rhodopsin, the number of photoreceptor cells was progressively reduced, because of retinal degeneration, but dark-adapted cAMP levels did not decline accordingly. P23H transgenic animals of both lines had higher levels of cAMP per photoreceptor cell count than control animals. This elevation was more pronounced as degeneration progressed. S334ter animals showed smaller cAMP elevation than P23H rats at a similar stage of retinal degeneration, but at a point when S334ter rats were undergoing rapid retinal degeneration, whereas in P23H rats retinal degeneration was slowing down. CONCLUSIONS: All three lines of transgenic rats carrying rhodopsin mutations show an increase in dark-adapted photoreceptor cAMP levels. A complex relationship exists between cAMP levels and the rate of cell death in the retina. Although initially higher levels of cAMP may promote cell survival and slow down retinal degeneration, ultimately, elevated cAMP levels may become toxic and may contribute to retinal cell death.


Subject(s)
Cyclic AMP/metabolism , Mutation , Photoreceptor Cells, Vertebrate/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Rhodopsin/genetics , Animals , Animals, Genetically Modified , Cell Count , Dark Adaptation , Disease Progression , Female , Immunohistochemistry , Male , Photoreceptor Cells, Vertebrate/pathology , Rats , Rats, Sprague-Dawley , Retinal Degeneration/pathology
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