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Frank P Stefano

Publications and source records attributed to Frank P Stefano.

5 recordsLinked to original sources
PubMed2006

ROM-1 potentiates photoreceptor specific membrane fusion processes.

Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin's non-glycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-1/P/rds complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes.

Animals↗
PubMed2006

Nonvisual arrestin oligomerization and cellular localization are regulated by inositol hexakisphosphate binding.

Interactions between arrestins and phosphoinositides have been reported to regulate multiple membrane-associated signaling and trafficking events including clathrin-mediated endocytosis and light adaptation in Drosophila. Arrestins have been proposed to have nuclear and cytosolic functions as well, although the ligand dependence of these functions has not been investigated. Here we characterize the structural, molecular, and cellular interactions between arrestin-2 and inositol hexakisphosphate (inositol 1,2,3,4,5,6-hexakisphosphate (IP(6))). The crystal structure of the arrestin-2.IP(6) complex was solved to 2.9 A with crystal lattice contacts suggesting two sites on a protein monomer mediating IP(6) binding. Mutagenesis coupled to isothermal titration calorimetry and tritiated IP(6) binding assays confirmed two-site binding with a low affinity IP(6)-binding site in the N-domain and a high affinity site in the C-domain. Native gel electrophoresis, gel filtration, and analytical ultracentrifugation demonstrated the ability of IP(6) to promote arrestin-2 oligomerization via the two crystallographically defined ligand-binding locations. In addition, analysis in mammalian cells revealed that arrestin-2 not only undergoes homo-oligomerization, but it can also hetero-oligomerize with arrestin-3 in a manner that depends on IP(6)-binding sites. Mutation of either IP(6)-binding site in arrestin-2 disrupted oligomerization while interactions with known binding partners including clathrin, AP-2, and ERK2 were maintained. Subcellular localization studies showed that arrestin-2 oligomers are primarily cytoplasmic, whereas arrestin-2 monomers displayed increased nuclear localization. Thus, by promoting cytosolic oligomerization, IP(6) binding is proposed to be a negative regulator of interactions of arrestin with plasma membrane and nuclear signaling proteins.

Animals↗
PubMed2004

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling.

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.

Amino Acid Sequence↗
PubMed2002

Heterologous expression of WT and mutant photoreceptor peripherin/rds in Madin Darby canine kidney cells: an assessment of fusogenic function.

Peripherin/rds is proposed to function as a fusion protein within the rod outer segment and a fusion domain has been mapped to amino acids 311-325 within the C-terminus. To map regions within peripherin/rds required for membrane fusion a series of C-terminal mutants was analyzed. Madin Darby canine kidney cells were transiently transfected with an Xpress or FLAG epitope tagged peripherin/rds (wt) and three mutants of peripherin/rds. The mutants selected were a P296T mutant (replacement of the proline at position 296 with a threonine) and two C-terminal deletion mutants (one lacking the terminal 10 amino acids, Delta10 and one lacking the terminal 50 amino acids, Delta50). The wt protein, the P296T and Delta10 mutants were detected on SDS-PAGE as 84 kDa dimers, that resolved into 38-42 kDa monomers under reducing conditions. The Delta50 mutant showed a slightly increased mobility. The cellular localization of mutants differed from that of wt peripherin/rds. The wt Xpress-human and wt FLAG-bovine peripherin/rds were localized to both intracellular and plasma membranes. In contrast, the C-terminal deletion mutants were localized only to the intracellular membrane. The P296T mutant presented a still different pattern: initially the protein localized to intracellular membranes. Upon confluence, however, the localization appeared to become predominantly plasma membrane. To assess the fusion activity of the proteins, the cell membranes were fractionated using sucrose density gradient centrifugation and the various fractions identified based on immunoreactivity in Western blot analysis with Golgi (anti-rab 6) or plasma membrane (anti-ZO-3) specific marker proteins. All membrane fractions were assayed for fusion with ROS plasma membrane vesicles. The plasma membrane enriched fractions (isolated at densities of 1.08 and 1.125 g ml(-1)) containing tagged peripherin/rds and the Delta10 mutant promoted membrane fusion with ROS plasma membrane vesicles. In contrast, fusion was not detected with plasma membrane vesicles from mock-transfected cells or the Delta50 peripherin/rds deletion mutant. Fusion was enhanced in a less dense fraction enriched in the P296T mutant (isolated from the 1.04/1.02 interface) relative to wt. Fusion was dependent on the presence of peripherin/rds in the membranes and could be inhibited with trypsinolysis and competition studies with the bovine fusion peptide, PP-5. Peptide competition suggests that the fusion domain of human peripherin/rds is most likely identical to that characterized in bovine and corresponds to amino acid residues 312-326. The C-terminal deletion mutants have allowed us to predict the minimal region of the C-terminus necessary for fusion to include residues starting at number 335. In addition a second region important in the formation of a fusion competent peripherin/rds has been mapped to a region upstream of the fusion peptide domain.

Animals↗