Abstract
The G protein beta subunit, Gbeta5, is predominantly expressed in the central nervous system. In rodent brain, Gbeta5 is expressed as a protein with an apparent molecular mass of 39,000 daltons (39 kDa). We have identified an additional Gbeta5 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 Gbeta5 (Gbeta5L) 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 Gbeta5 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 Gbeta5L protein co-purified with rod outer segment membranes. Incubation of rod outer segment membranes with the nonhydrolyzable guanine nucleotide, GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), which released the Gbeta subunit of transducin (Gbeta1), failed to remove Gbeta5L. The 39-kDa Gbeta5 protein displayed differential association with retinal and brain membranes. In the retina, Gbeta5 was present as a soluble protein and was undetectable in the membrane fraction, whereas in the brain approximately 70% of Gbeta5 was associated with cellular membranes. In transient COS-7 cell expression experiments, Gbeta5L formed functional Gbetagamma dimers and Galphabetagamma heterotrimers, and activated phosphoinositide-specific phospholipase Cbeta2 in a manner indistinguishable from the 39-kDa Gbeta5 protein. The cloning of the retinal-specific Gbeta5L cDNA suggests the existence of potentially novel G protein-mediated signaling cascades in photoreception.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U69145
Identification and cDNA Cloning of a Retinal-specific Form of G5—We have reported previously that the G protein  subunit, G5, is expressed predominantly in the central nervous system
The G␥ dimers of heterotrimeric G proteins have emerged in recent years as important regulators of ion channels, adenylyl cyclases, phosphoinositol-specific phospholipases, G protein-coupled receptor kinases, and mitogen-activated protein kinase cascades [2, 3]
Summary
One clone was identified that contained approximately 8 kb of 5Ј untranslated DNA This 8-kb region was cloned into pBluescript SKII and partially sequenced by primer walking. For Northern analysis, 40 g of total RNA, prepared from mouse tissues using the guanidine thiocyanate-phenol method (TriZol reagent, Life Technologies, Inc.), was fractionated on formaldehyde-agarose gels, transferred to Magna NT nylon membranes (MSI) and probed with an oligonucleotide specific for the predicted 5Ј-untranslated region of the long form of G5 (5Ј-CTTGGCTGAGATGAGGCTAAAGGACAGAAAAGAATGAACAGGG-3Ј). The level of inositol phosphates was determined 25 min after addition of 10 mM LiCl. For Western analysis, 1 ϫ 105 transiently transfected cells were scraped into 60 l of lysis buffer (50 mM HEPES, pH 7.4, 1 mM EGTA, 10 mM DTT, 0.2 TIU/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride), and membranes were pelleted at 15,000 ϫ g for 30 min in an Eppendorf centrifuge. Proteins were separated on 10% SDS-PAGE gels, transferred to nitrocellulose, and visualized as described above
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