Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos.
SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.
Kontrogianni-Konstantopoulos
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Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza Houston, Texas, USA.
Flytzanis
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CN
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002451:Cell Compartmentation; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D008845:Microinjections; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D012617:Sea Urchins / Q000166:cytology / Q000196:embryology* / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004527:Egg Proteins; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors