A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR ␣ subtypes from spotted seatrout A LTHOUGH THE IMPORTANCE of rapid (i.e. nonclassical) steroid actions initiated at the cell surface through binding to steroid membrane receptors has become more widely accepted within the past few years, details of the initial steroid-mediated events, including the identities of the steroid membrane receptors and their mechanisms of action, remain unclear and are surrounded by controversy (1-3). There is clear evidence that a variety of receptor proteins are involved in initiating these nonclassical steroid actions in different cell models, including nuclear steroid receptors or nuclear steroid receptor-like forms (1, 2, 4), receptors for other ligands that also bind steroids (2, 5), and unidentified receptors with different characteristics from those of any known receptors (2, 6). Recently, a novel cDNA was discovered in spotted seatrout ovaries that has several characteristics of the progestin membrane receptor (mPR) mediating progestin induction of oocyte maturation in this species by a nongenomic mechanism (7). The seatrout cDNA (st-mPR␣) encodes a 40 kDa protein, which has seven transmembrane domains, and receptor activation alters pertussis toxin-sensitive adenylyl cyclase activity, both of which suggest stmPR␣ is a G protein-coupled receptor (GPCR) or GPCR-like protein (7). More than 20 closely related genes have been cloned from other vertebrate species, including three mPR subtypes in humans, named ␣, ␤, and ␥, which show high levels of expression in human reproductive, brain, and kidney tissues, respectively (8). The identification of a new class of putative steroid receptors, unrelated to nuclear steroid receptors, but instead related to GPCRs, provides a plausible explanation of how steroids can initiate rapid hormonal responses in target cells by activating receptors on the cell surface. There has been broad recognition of the potential significance of these findings (1, 9, 10) and also an extensive research effort to determine the distribution, hormonal regulation, and biological roles of the mPRs in various vertebrate models (11-16). However, critical information is still lacking on several key features of mPRs essential for clearly establishing this proposed alternative model of steroid action and for understanding its likely evolutionary origins.The st-mPR␣ protein has been localized to the plasma membrane of seatrout oocytes (7), but progestin binding and activation of signal transduction pathways in the plasma membranes of cells transfected with the st-mPR␣ and human mPRs remain to be demonstrated. To date, progestin binding has only First Published Online November 9, 2006Abbreviations: GPCR, G protein-coupled receptor; HLY3, hemolysin 3; hu-mPR␣, human membrane progestin receptor ␣; MMD, monocyte to macrophage differentiation protein; mPR, membrane progestin rece...

Often the definition of orthology is incorrectly interpreted as a prediction of proteins that are functionally equivalent across species, while in fact it only defines the existence of a common ancestor for a gene in different species. However, it has been demonstrated that orthologs often reveal significant functional similarity. Therefore, the quality of the orthology prediction is an important factor in the transfer of functional annotations (and other related information). To identify protein pairs with the highest possible functional similarity, it is important to qualify ortholog identification methods.

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G 2 /M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G 2 /M transition. However, the FHA domain blocked ␤-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.Type 1 protein phosphatases (PP1) 1 belong to the PPP family of Ser/Thr protein phosphatases and regulate diverse cellular processes such as transcription, pre-mRNA splicing, intracellular transport, and metabolism (1-3). They consist of a single catalytic subunit (PP1 C ) and one or two regulatory subunits. The regulatory subunits act as substrate specifiers and anchor the holoenzymes in specific cell compartments in close vicinity to their substrates. In addition, the regulatory subunits mediate the control of the holoenzymes by hormones and growth factors through interaction with allosteric effectors or through phosphorylation by specific protein kinases. It has been estimated that mammalian cells contain tens of different regulatory proteins of PP1 (4). Altogether about 20 of these have already been characterized and cloned, including the glycogenbinding G-subunits, the myosin-binding M-subunits, the cytosolic regulator inhibitor-1, and the nuclear RNA-binding protein NIPP1 (1-3). Recent investigations have revealed that these regulatory proteins have multiple points of interaction with PP1 C , including a common phosphatase binding motif with the consensus sequence RVXF (5-10). In addition, most regulatory subunits contain domains that mediate the binding to substrates (e.g. myosin for the M-subunits) and/or a subcellular structure (e.g. glycogen for the G-subunits) to which the substrates are bound.In nuclear extracts, NIPP1 (39 kDa) is present as an inactive complex with PP1 C , termed PP1N NIPP1 (11). This heterodimer can be activated by phosphorylation of up to 4 Ser/Thr residues in the central domain of NIPP1 by protein kinases A and CK2 (12), which disrupts its interaction with PP1 C via the RVXF motif without dis...