Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/ peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.The outer segments (OSs) 1 of rod and cone photoreceptor cells act as detectors of visible light for the initial stages of the visual process and are vital for normal vertebrate vision. The OS is an architecturally complex organelle. It contains hundreds of precisely stacked membranous disks that undergo a polarized renewal process; a complete turnover of OS membrane protein occurs approximately once every 10 days in primate rod cells (1). Although the renewal process and OS stability are essential for photoreceptor viability, their underlying molecular bases remain largely undefined. Evidence from several laboratories has implicated the integral membrane protein peripherin/rds in OS morphogenesis and the renewal process (2-4). It is present in all vertebrate rod and cone photoreceptors examined to date and causes a variety of progressive retinal diseases in humans when defective (5).Despite continued interest ...
Under normal culturing conditions, the T47D human breast cancer cell line expresses progesterone receptor constitutively and is responsive to estrogen. Because the tumor suppressor protein p53 plays a central role in determining genetic stability and cell proliferation, we have examined the effects of 17-estradiol, the synthetic progestin R5020, and the antiprogestin RU486 on the levels of this protein in T47D cells. Western blot analysis of cellular extracts, performed with a monoclonal antibody capable of quantitatively supershifting a specific p53-p53 response element complex in a gel mobility shift assay, detected a single immunoreactive band representing p53. When cells were grown for 4 -5 days in culture medium containing charcoal-treated fetal calf serum, p53 levels declined to 10% of the level seen in the control (no charcoal treatment) group. Supplementation of culture medium containing charcoal-treated calf serum with 0.1-1 nM 17-estradiol restored p53 to its normal levels. A 4-day treatment of cells with R5020 or RU486 lowered the p53 levels in cells grown in normal culturing conditions to 15 and 30% of control levels, respectively. R5020 and RU486 treatments also caused down-regulation and/or hyperphosphorylation of the progesterone receptor, which correlated with the downregulation of p53. These observations indicate that in T47D cells, p53 is up-regulated by estradiol while R5020 down-regulates this protein. Since estradiol is known to promote cell proliferation, the induction of p53 observed in this study leads us to propose that estradiol stimulates p53 to regulate proliferation of T47D cells in culture.The development, growth, and differentiation of human breast is under the influence of a number of hormones including the sex steroids, estradiol (E 2 ) 1 and progesterone. In cases where the breast tissue transforms into a tumorous entity, it often continues to respond to circulating levels of these hormones provided it expresses receptors for the corresponding hormone. While hormone-insensitive malignancies do not appear to express functional receptors, hormone-sensitive cancers may overexpress progesterone receptor (PR) and estradiol receptor (McGuire, 1978). Treatment of breast cancers with hormones or antihormones presumes the presence of functional receptors, which via activation or inactivation of receptors mediate regression of cancerous tissues. Although breast cancers may initially respond to endocrine therapy, the tissue can transform into a hormone-insensitive entity. The mechanisms underlying the evolution of hormone-sensitive tumors to hormone-insensitive states are not known (Horwitz, 1994). It is, therefore, crucial to determine whether treatment with hormones and/or antihormones might affect a shift toward a progressively more malignant state of breast cancer. The biological activity of the tumor suppressor protein p53 is associated with suppression of cell growth. It is now widely recognized that p53 may be the most frequently mutated protein in human cancers (Oren, 1992). Beca...
SummaryVertebrate vision requires photon absorption by photoreceptor outer segments (OSs), structurally elaborate membranous organelles derived from non-motile sensory cilia. The structure and function of OSs depends on a precise stacking of hundreds of membranous disks. Each disk is fully (as in rods) or partially (as in cones) bounded by a rim, at which the membrane is distorted into an energetically unfavorable high-curvature bend; however, the mechanism(s) underlying disk rim structure is (are) not established. Here, we demonstrate that the intrinsically disordered cytoplasmic C-terminus of the photoreceptor tetraspanin peripherin-2/rds (P/rds) can directly generate membrane curvature. A P/rds C-terminal domain and a peptide mimetic of an amphipathic helix contained within it each generated curvature in liposomes with a composition similar to that of OS disks and in liposomes generated from native OS lipids. Association of the C-terminal domain with liposomes required conical phospholipids, and was promoted by membrane curvature and anionic surface charge, results suggesting that the P/rds C-terminal amphipathic helix can partition into the cytosolic membrane leaflet to generate curvature by a hydrophobic insertion (wedging) mechanism. This activity was evidenced in full-length P/rds by its induction of small-diameter tubulovesicular membrane foci in cultured cells. In sum, the findings suggest that curvature generation by the P/rds Cterminus contributes to the distinctive structure of OS disk rims, and provide insight into how inherited defects in P/rds can disrupt organelle structure to cause retinal disease. They also raise the possibility that tethered amphipathic helices can function for shaping cellular membranes more generally.
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