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A Novel Germ Line-specific Gene of the Phosducin-like Protein (PhLP) Family

A MEIOTIC FUNCTION CONSERVED FROM YEAST TO MICE*
Open AccessPublished:January 17, 2003DOI:https://doi.org/10.1074/jbc.M207434200
      We identified a new member of the phosducin-like (PhLP) protein family that is predominantly, if not exclusively, expressed in male and female germ cells. In situ analysis on testis sections and analysis of purified spermatogenic cell fractions evidenced a stage-specific expression with high levels of RNA and protein in pachytene spermatocytes and round spermatids. Three mRNA species were detected, which correspond to different polyadenylation sites and vary in abundance during germ cell maturation. Only low levels of RNA were detected in whole ovary extracts, but expression of the protein became detectable within hours after hormonal induction of superovulation. The gene (Mgcphlp) is located on mouse chromosome 5 in the immediate vicinity of the Clock locus. The predicted amino acid sequence shows extensive similarities not only with the known mammalian PhLP proteins but also with the yeast phosducin-like protein Plp2, required for the production and growth of haploid cells. Expression of the murine protein was found to complement the defect of a yeastplp2Δ mutant. We propose that MgcPhLP/Plp2 proteins exert a function in germ cell maturation that is conserved from yeast to mammals.
      PhLP
      phosducin-like protein
      GST
      glutathione S-transferase
      RACE
      rapid amplification of cDNA ends
      RT
      reverse transcriptase
      EST
      expressed sequence tag
      Phosducin, a protein highly expressed in the retina and pineal gland, has been considered as playing a role in retinal phototransduction by interacting with the βγ subunits of G proteins and thereby modulating their signaling functions (
      • Lee R.H.
      • Whelan J.P.
      • Lolley R.N.
      • McGinnis J.F.
      ). A partially similar, widely expressed phosducin-like protein (PhLP)1 has been identified, which also inhibits Gβγ function (
      • Schroder S.
      • Lohse M.J.
      ,
      • Thibault C.
      • Sganga M.W.
      • Miles M.F.
      ). Two related genes identified in the yeast Saccharomyces cerevisiae were designated PLP1 and PLP2 (
      • Flanary P.L.
      • DiBello P.R.
      • Estrada P.
      • Dohlman H.G.
      ). The Plp1 protein was shown to bind efficiently the Gβγ subunits. Binding of Plp2 was also evidenced but with a lesser affinity. On the other hand, genetic analysis evidenced the role of Plp2 in the generation of viable haploid cells.
      The mammalian phosducin and phosducin-like proteins are expressed ubiquitously (
      • Danner S.
      • Lohse M.J.
      ). Whether tissue-specific homologues exist remains an open question. In the course of screening a mouse testis cDNA library, we have identified a novel germ cell-specific phosducin-like protein, designated MgcPhLP (for “mouse germ cell-specific phosducin-like protein”), which exhibits significant similarities to both the mouse phosducin and phosducin-like proteins. Expression is strictly restricted to the male and female germ cells in a regulated manner depending on the stage of germ cell maturation. The murine gene complemented the defect of a yeast plp2Δ mutant, suggesting an evolutionarily conserved function in meiotic and/or post-meiotic cells.

      DISCUSSION

      Phosducins are regulators of G protein activity in the retina, and the phosducin-like proteins are considered to be potential ubiquitous regulators of Gβγ signaling. We describe in this report a novel phosducin-like mRNA specific of the meiotic and post-meiotic germ cells, which shows significant amino acid sequence similarities to the phosducin and phosducin-like proteins of various species. Searching existing EST clones from both mouse and human suggested germ cell specificity, because related sequences were found in EST libraries from testes but not from other tissues. In the mouse, expression ofMgcphlp was at least predominantly observed in male and female germ cells, in both sexes at the meiotic and post-meiotic stages. It must be taken into account, however, that a detailedin situ analysis could not be performed on every possible tissue, and therefore we cannot exclude the possibility that the gene might be expressed in a minor cell population and/or only during a limited physiological or developmental period (as is in fact the case in the ovary). In the male gonad, three Mgcphlp RNA isoforms were identified at different stages of differentiation. These three specific mRNAs correspond to different polyadenylation sites in the locus. It is interesting to note the presence in the vicinity of the polyadenylation sites in genomic DNA of AU-rich sequences similar to the sequences described as cytoplasmic polyadenylation elements mediating polyadenylation and translation of messages during the oocyte release from the meiotic block at ovulation and prior to the activation of zygotic genome at the two cell stage (
      • Oh B.
      • Hwang S.
      • McLaughlin J.
      • Solter D.
      • Knowles B.B.
      ,
      • Stutz A.
      • Conne B.
      • Huarte J.
      • Gubler P.
      • Volkel V.
      • Flandin P.
      • Vassalli J.D.
      ). The presence of stage-specific isoforms of the Mgcphlp message may thus reflect a translational control during meiotic and post-meiotic maturation. The conclusions of RNA analysis were confirmed by direct determination of the protein by polyclonal antibodies specific for the mouse protein. This was especially informative in the ovary, in which expression is normally limited to the small number of oocytes undergoing meiosis but could readily be evidenced after hormonal stimulation leading to superovulation.
      As is the case for the other phosducin-related proteins, the function of the protein at the molecular level remains largely to be established. A possibly significant feature in this respect is its association with at least one of the proteins of the 14-3-3 family. Binding may be mediated by the RSSVP motif (amino acids 119–123, Fig. 1), which resembles the sites of interaction identified in other 14-3-3-binding proteins (
      • Aitken A.
      ). 14-3-3 binds phosphorylated serine residues in a number of proteins active in signal transduction. In retinal photoreceptors, 14-3-3 is considered as regulating the binding of phosducin to Gβγ by sequestering the phosphorylated phosducin molecules and blocking their binding. 14-3-3 was also recently shown to interact in the brain with a phosducin-like protein (
      • Garzon J.
      • Rodriguez-Diaz M.
      • Lopez-Fando A.
      • Garcia-Espana A.
      • Sanchez-Blazquez P.
      ,
      • Nakano K.
      • Chen J.
      • Tarr G.E.
      • Yoshida T.
      • Flynn J.M.
      • Bitensky M.W.
      ). The specificity of 14-3-3 binding and its relationship with the phosphorylation of serine residues in MgcPhLP are currently being studied.
      Two phosducin-related genes were recently described in yeast,PLP1 and PLP2 (
      • Flanary P.L.
      • DiBello P.R.
      • Estrada P.
      • Dohlman H.G.
      ). MgcPhLP displays a greater amino acid similarity with PLP2 than withPLP1. The inability of a plp2Δ mutant to generate viable haploid products was successfully complemented by transfer of the mouse gene. Regarding the function of the yeast gene, published data (
      • Flanary P.L.
      • DiBello P.R.
      • Estrada P.
      • Dohlman H.G.
      ) have left two possibilities open; the Plp2 protein could either be necessary for growth in general or specifically required for the generation of haploid products, either during or after meiosis. The observation that haploid clones bearing theplp2Δ mutation could not be grown from a sporulating culture is compatible with both interpretations, thus making it impossible to evaluate the phenotype of the diploid homozygous mutant. Our data, in fact, rather favor the hypothesis of a meiotic function. We observed, upon long term growth of several of the complemented haploid strains, a loss of the murine gene that did not impair their growth ability. A meiotic function of the yeast gene is consistent with the restricted meiotic and post-meiotic expression ofMgcphlp in the mouse.
      Mgcphlp is included in the 204-kb DNA fragment of theClock locus (GenBankTM accession numberAF146795). Taking into account that phosducin, which is expressed abundantly in the retina, has been considered to be involved in signal phototransduction cascades, one might speculate that Mgcphlpexpression could be part of the same type of signaling cascade initiated by dark/light stimuli. In the mouse, the ovulation cycle is known to be dependent on light periodicity, and circadian periods have been reported for the ovarian melatonin and rhythm of cAMP accumulation. Increase in Mgcphlp expression within hours after induction of superovulation by human chorionic gonadotropin injection clearly points to hormonal regulation. It is clear, however, that beyond such speculations, the function of the mouse protein in germ cell differentiation will require the use of site-directed and/or temporally controlled mutagenesis technologies.

      Acknowledgments

      We thank Rob Arkowitz for help in the design of yeast experiments and Yan Fantei and Mireille Cutajar for skilled technical help.

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