The reproductive importance of P-type ATPases.

P-type ATPases are integral membrane proteins that use the free energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients to support a variety of cellular processes. They have eight signature motifs, eight or ten transmembrane domains, highly conserved phosphorylation and ATP-binding sites, and similar hydropathic profiles. This review summarizes recent insights in the relationship of P-type ATPases to successful reproduction, and the hormone dependence of some family members. Because protein topology is central to understanding the pump action of this family of enzymes, this review also describes the dramatic change in the primary structure of one family member that may mediate transcription in the uterus.

Oviductin (Muc9) is expressed in rabbit endocervix.

While isolating and characterizing cervical mucin glycoproteins, oviductin (Muc9) was identified in the rabbit endocervix. Following tissue homogenization, endocervical proteins were fractionated by exclusion chromatography (Sepharose CL-4B). High molecular weight components of the void volume were resolved by density gradient centrifugation using cesium bromide and dissociative conditions (4 M guanidinium chloride). High density fractions (rho = 1.40 - 1.56 g/ml) were deglycosylated with anhydrous trifluoromethane sulfonic acid and sent to Harvard Microchemistry where in situ digestion and tryptic peptide separation were performed. Out of an HPLC map, microsequence (KLIMGFPTYGR) from peak 51 was 100% identical to mouse oviductin, and microsequence (KSTGHNFPLP) from peak 70 was 90% identical to hamster oviductin. Temporal expression of oviductin transcripts (2.4-kilobase) was negligible during the first three months of postnatal cervical differentiation. Transcripts were minimally detectable in the cervices of 4-month-old juveniles. Strong expression in the endocervices of adults was eliminated by ovariectomy and restored by estrogen treatment. The presence of oviductin in the rabbit endocervix indicates this glycoprotein may have multiple functions, and it can no longer be considered oviduct-specific.

Cloning and characterization of an atypical Type IV P-type ATPase that binds to the RING motif of RUSH transcription factors.

RUSH proteins are SWI/SNF-related transcription factors with RING finger signatures near their COOH termini. Long suspected of mediating protein-protein interactions, the RING motif was used to clone a binding partner. The RING finger binding protein (RFBP) is a Type IV P-type ATPase, a putative phospholipid pump, with conserved sequences for two loop segments, an ATP-binding site, a phosphorylation domain, and transmembrane passes potentially involved in substrate binding and translocation. However, RFBP differs from all other Type IV P-type ATPases in three ways. It has only three of four highly conserved NH(2)-terminal transmembrane passes, it is located in the inner nuclear membrane, and it binds the RING domain. Topographically the orientation of the adjacent hydrophilic domains and the determinants of transport specificity are altered. As a result, the small, hydrophilic loop extends into the perinuclear space that is contiguous with the lumen of the endoplasmic reticulum. The large, conformationally flexible loop extends into the nucleoplasm to contact euchromatin. Competitive reverse transcriptase-polymerase chain reaction and high performance liquid chromatography analysis revealed that endometrial RFBP mRNA expression is hormonally regulated. The physical association of a hormone-dependent RING finger-binding protein with transcriptionally active chromatin supports the speculation that RFBP plays a role in the subnuclear trafficking of transcription factors with RING motifs.

Uteroglobin gene transcription: what's the RUSH?

Prolactin enhances progesterone-dependent transcription of the rabbit uteroglobin gene. RUSH transcription factors are implicated in the signal transduction pathway. The RUSH acronym identifies key features of these nuclear phosphoproteins, that is, RING-finger motif, binds the uteroglobin promoter, structurally related to the SWI/SNF family of transcription factors, and helicase-like. Cloned by recognition site screening, RUSH proteins bind to an 85-bp region (-170/-85) of the uteroglobin promoter that was subsequently identified as a novel prolactin-responsive region by promoter deletion analysis. Gel shift and linker-scanning assays further reduced the RUSH target site to -160/-110. A hexameric core of MCWTDK was identified as the RUSH-specific DNA-binding site (-126/-121) by CASTing. This site overlaps authentic HNF3 beta and OCT-1 binding sites. A unique Type IV P-type ATPase that is embedded in the inner nuclear membrane binds the RING domain of RUSH. The conformationally flexible loop portion of this RING-finger binding protein (RFBP) extends into the nucleoplasm to contact euchromatin. The physical association of RFBP with transcriptionally active chromatin supports the speculation that RFBP targets RUSH transcription factors to the active uteroglobin promoter.