RESUMO
The novel rhomboid-like protein RHBDD2 is distantly related to rhomboid proteins, a group of highly specialized membrane-bound proteases that catalyze regulated intramembrane proteolysis. In retina, RHBDD2 is expressed from embryonic stages to adulthood, and its levels show age-dependent changes. RHBDD2 is distinctly abundant in the perinuclear region of cells, and it localizes to their Golgi. A glycine zipper motif present in one of the transmembrane domains of RHBDD2 is important for its packing into the Golgi membranes. Its deletion causes dislodgment of RHBDD2 from the Golgi. A specific antibody against RHBDD2 recognizes two forms of the protein, one with low (39 kDa; RHBDD2(L)) and the other with high (117 kDa; RHBDD2H) molecular masses in mouse retinal extracts. RHBDD2(L) seems to be ubiquitously expressed in all retinal cells. In contrast, RHBDD2H seems to be present only in the outer segments of cone photoreceptors and may correspond to a homotrimer of RHBDD2(L). This protein consistently co-localizes with S- and M-types of cone opsins. We identified a homozygous mutation in the human RHBDD2 gene, R85H, that co-segregates with disease in affected members of a family with autosomal recessive retinitis pigmentosa. Our findings suggest that the RHBDD2 protein plays important roles in the development and normal function of the retina.
Assuntos
Endopeptidases/biossíntese , Endopeptidases/fisiologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Retina/metabolismo , Retinose Pigmentar/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Feminino , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Glicina/química , Complexo de Golgi/metabolismo , Células HEK293 , Homozigoto , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Opsinas/química , Gravidez , Prenhez , Retina/embriologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The large tumor suppressor gene (Lats1) encodes a protein kinase that is highly conserved from fly to human, and plays a crucial role in the prevention of tumor formation by controlling mitosis progression. We have found that in addition to the previously isolated 7.5 kb long form of Lats1 (Lats1L) mRNA, a less abundant, shorter, 3.4 kb primary transcript (Lats1S) also is expressed in the vertebrate retina. Compared to Lats1L, the sequence of Lats1S mRNA has a deletion of exons 6, 7, and 8 that corresponds to 792 bp of the open reading frame. Thus, 264 aa of the C-terminal region of the long transcript are missing in the Lats1S protein. The encoded truncated protein lacks four of eleven conserved kinase domains and the C-terminus. Our results suggest that the 3.4 kb transcript is a splice variant of the 7.5 kb transcript. We have found direct evidence that both the retinal 7.5 and 3.4 kb mRNAs are translated into 170 kDa and 120 kDa proteins, respectively. The expression of both isoforms in vertebrate cells raises the possibility that these Lats1 proteins may act as negative key regulators of the cell cycle, each of them performing a unique role.
Assuntos
Cães/genética , Proteínas Serina-Treonina Quinases/genética , Retina/metabolismo , Deleção de Sequência/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Humanos , Immunoblotting , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
PURPOSE: A functional protein is required for structure/function analysis of cone photoreceptor cGMP-phosphodiesterase alpha' subunit (PDEalpha'). The purpose of this study was to express enzymatically active PDEalpha'. METHODS: Three expression vectors were constructed for transient and stable expression of PDEalpha': pC57 (transient) was obtained by subcloning bovine PDEalpha' cDNA into the pCIS2 expression vector; pNC57 (stable) was constructed by inserting the neo gene controlled by the mouse phosphoglycerate kinase-1 gene promoter into the pC57 vector; and pFC57 (transient) was generated by fusing the sequence encoding the FLAG peptide to the 5' end of the coding region of PDEalpha' cDNA. The recombinant plasmid DNAs were introduced into HEK293, CHO, or Y79 retinoblastoma cells using the calcium phosphate-mediated transfection procedure or lipofectamin. Northern and western blot hybridizations were used for RNA and protein analysis, respectively. RESULTS: Northern blots of both HEK293- and CHO-transfected cells showed strong expression of a 3 kb transcript corresponding to PDEalpha'. cGMP-PDE activity measured in homogenates of transiently and stably transfected cells ranged between 1.5 and 2.2 nmol cGMP hydrolyzed/min x mg total protein, a level of PDE activity slightly greater than that previously reported for the individual rod-photoreceptor PDE subunits transiently-expressed in HEK293 cells. Western blots of these cell homogenates showed a low level of expressed PDEalpha'. Transfection of Y79 retinoblastoma cells, that have been shown to express rod and cone PDEs endogenously, with the construct containing cone PDEa' cDNA fused to the FLAG peptide resulted in a protein with no enzymatic activity. CONCLUSIONS: Our results demonstrate that both HEK293 and CHO cells are capable of expressing functionally active cone PDEalpha'. High level of mRNA transcription and relatively low protein synthesis efficiency indicates the presence of a post-transcriptional control mechanism regulating overall expression of PDEalpha' in HEK293 and CHO cells.