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 embryo s, larvae, and adult tissues of sea urchin. During embryonic development, t wo receptor isoforms are produced via alternative splicing. One exhibits th e typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs enc oding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression o f the tagged isoforms in S. purpuratus embryos showed that the exogenous Sp SHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length i soform is predominantly localized in the nucleus, whereas two cell division s later (16-cells) protein accumulations are detected in both the nucleus a nd 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 int racellular distribution of two receptor mutants, one having a deletion with in the DBD (DeltaP) and the other a truncation of the C-terminal F-domain ( DeltaF), revealed that DeltaP is localized similarly to full-length recepto r, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splic e variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1- elementas monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR 1 poorly. These results suggest that the receptor's putative LBD is respons ible for the differential subcellular localization of the two natural SpSHR 2-isoforms in early development. Mol. Reprod. Dev. 60: 147-157, 2001. (C) 2 001 Wiley-Liss, Inc.