Molecular Cloning and Characterization of the G Protein γ Subunit of Cone Photoreceptors

Ong, Olivia C.; Yamane, Harvey; Phan, Kim B.; Fong, Henry K. W.; Bok, Dean; Lee, Rehwa H.; Fung, Bernard K.-K.

doi:10.1074/jbc.270.15.8495

Cited by 81 publications

(54 citation statements)

References 55 publications

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“…In addition, a novel cone-specific G␥ subunit (G␥ 8 ) has been reported recently (35). Preliminary immunohistochemical analysis of mouse retinal sections with antiserum CT215 indicates that G␤ 5 immunoreactivity is present in the plexiform layers, similar to the reported distribution for bovine G␥ 7 .…”

Section: Discussionmentioning

confidence: 52%

A Novel Form of the G Protein β Subunit Gβ5 Is Specifically Expressed in the Vertebrate Retina

Watson

Aragay

Slepak

et al. 1996

Journal of Biological Chemistry

131

115

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The G protein ␤ subunit, G␤ 5 , is predominantly expressed in the central nervous system. In rodent brain, G␤ 5 is expressed as a protein with an apparent molecular mass of 39,000 daltons (39 kDa). We have identified an additional G␤ 5 immunoreactive protein of apparent size 44 kDa in the vertebrate retina. Molecular cloning and sequencing of polymerase chain reaction products revealed that the cDNA encoding the larger species of G␤ 5 (G␤ 5L ) was identical to the shorter form with the addition of 126 base pairs of 5 DNA sequence potentially encoding an in-frame 42-amino acid extension. Sequencing of mouse G␤ 5 genomic clones demonstrated that the 126-base pair of retinal-specific coding material is derived from a hitherto undetected 5 exon. During sucrose density gradient fractionation of bovine retinas, the 44-kDa G␤ 5L protein co-purified with rod outer segment membranes. Incubation of rod outer segment membranes with the nonhydrolyzable guanine nucleotide, GTP␥S (guanosine 5-3-O-(thio)triphosphate), which released the G␤ subunit of transducin (G␤ 1 ), failed to remove G␤ 5L . The 39-kDa G␤ 5 protein displayed differential association with retinal and brain membranes. In the retina, G␤ 5 was present as a soluble protein and was undetectable in the membrane fraction, whereas in the brain approximately 70% of G␤ 5 was associated with cellular membranes. In transient COS-7 cell expression experiments, G␤ 5L formed functional G␤␥ dimers and G␣␤␥ heterotrimers, and activated phosphoinositide-specific phospholipase C␤ 2 in a manner indistinguishable from the 39-kDa G␤ 5 protein. The cloning of the retinal-specific G␤ 5L cDNA suggests the existence of potentially novel G protein-mediated signaling cascades in photoreception.In eukaryotic cells, a family of signal-transducing guaninenucleotide binding proteins (G proteins) 1 orchestrates many physiological processes by coupling activated cell surface receptors to intracellular second messenger systems. G protein-coupled receptors (GPCRs), which possess a stereotypical seventransmembrane-spanning domain architecture, bind and mediate the signaling of a variety of molecules, including hormones, neurotransmitters, odorants, and light. To date, several hundred GPCRs have been cloned or characterized (1). In contrast, the number of heterotrimeric G proteins, as well as the number of G protein-regulated effectors, is much more limited.G proteins are heterotrimeric, composed of ␣, ␤, and ␥ subunits (G␣, G␤, G␥). Activation of a GPCR by ligand binding stimulates the exchange of bound GDP for GTP on the G␣ subunit and results in the dissociation of the G␣ subunit from a tightly complexed G␤␥ dimer. The released G␣ and G␤␥ subunits in turn regulate the activity of effector proteins, the better characterized of which include cGMP phosphodiesterase, adenylyl cyclases, phosphoinositide-specific phospholipase C ␤ enzymes, and ion channels. In addition, the dimeric G␤␥ subunits are involved in GPCR desensitization by recruitment of receptor kinases to the plasma membrane, and in ...

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Section: Discussionmentioning

confidence: 52%

A Novel Form of the G Protein β Subunit Gβ5 Is Specifically Expressed in the Vertebrate Retina

Watson

Aragay

Slepak

et al. 1996

Journal of Biological Chemistry

131

115

View full text Add to dashboard Cite

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“…Given its presence in rods, the phosphoinositide pathway is most likely present in cones as well, thus raising questions about the identities of the corresponding PLC enzyme and G ␣ protein in cones. One G protein ␤-subunit (G ␤3 ), but two ␥-subunits (G ␥2 and G ␥8 ), have recently been identified in cone outer segments (55)(56)(57). Biochemical analysis suggests that, functionally, G ␥8 is likely the cone homolog of rod G ␥1 and coupled to cone transducin (G ␣t2 ) (57).…”

Section: Discussionmentioning

confidence: 99%

“…One G protein ␤-subunit (G ␤3 ), but two ␥-subunits (G ␥2 and G ␥8 ), have recently been identified in cone outer segments (55)(56)(57). Biochemical analysis suggests that, functionally, G ␥8 is likely the cone homolog of rod G ␥1 and coupled to cone transducin (G ␣t2 ) (57). Perhaps, then, G ␥2 is coupled to the yet-to-be-identified G q member in cone outer segments.…”

Section: Discussionmentioning

confidence: 99%

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments

Peng

Rhee

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (␤1-4, ␥1-2, and ␦1-2), we have found PLC␤4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the ␣-subunits of the G q family of G proteins, which are known to activate PLC␤4, have also demonstrated G ␣11 -like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLC␤4 and anti-G ␣11 antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by in situ hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G ␣11 or a G ␣11 -like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G ␣q (another G q family member), are present elsewhere in the retina.Visual transduction in the retina takes place in the outer segments of rod and cone photoreceptors and is known to involve a cGMP signaling cascade (see ref. 1 for a recent review). In this process, light isomerizes the visual pigment into an active form, which, via the G protein transducin, stimulates a cGMP-phosphodiesterase to lead to cGMP hydrolysis. In darkness, cytoplasmic cGMP binds to and opens cGMP-activated cation channels on the plasma membrane of the outer segment. These open channels sustain an inward dark current to keep the cell partially depolarized and maintain a steady release of glutamate from the synaptic terminal of the photoreceptor. In the light, the hydrolysis of cGMP leads to the closure of these channels, producing a membrane hyperpolarization as the light response and reducing the glutamate release from the cell.Over the years, however, there have also been reports of a phosphoinositide signaling pathway in the rod outer segment. In this pathway, the membrane phospholipid phosphatidylinositol-4,5-bisphosphate is hydrolyzed by phospholipase C (PLC) enzymes to release the second messengers inositol-1,4,5-trisphosphate and diacylglycerol, with the former leading to intracellular Ca 2ϩ release and the latter activating the enzyme protein kinase C (PKC). In rod outer segment preparations, phosph...

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“…Thus, ␤␥ dimers containing ␥ subunits from subfamily I such as ␥ 1 or ␥ 11 (and probably ␥ 8 , although it has not been studied extensively) seem to have unique signaling properties in that they are unable to activate type II adenylyl cyclase or other important effectors, such as phosphatidylinositol-(4,5)-3-kinase (Kerchner et al, 2004). The distribution of ␥ 1 is restricted to retinal rods (Fung, 1983), and the original report of ␥ 8 (termed ␥ c ) suggested that it was restricted to cone cells (Ong et al, 1995). However, the ␥ 11 subunit is widely expressed , and it now seems that ␥ 8 is also widely expressed (Downes and Gautam, 1999).…”

Section: Interaction Of G Proteinmentioning

confidence: 99%

Regions in the G Protein γ Subunit Important for Interaction with Receptors and Effectors

Myung

Lim²,

DeFilippo³

et al. 2005

Mol Pharmacol

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G␤␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␤␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the ␤ 1 subunit, G␤␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-␤, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C-␤ isoform and inhibiting type I adenyl cyclase. In contrast, ␤␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.

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Molecular Cloning and Characterization of the G Protein γ Subunit of Cone Photoreceptors

Cited by 81 publications

References 55 publications

A Novel Form of the G Protein β Subunit Gβ5 Is Specifically Expressed in the Vertebrate Retina

A Novel Form of the G Protein β Subunit Gβ5 Is Specifically Expressed in the Vertebrate Retina

Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments

Regions in the G Protein γ Subunit Important for Interaction with Receptors and Effectors

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