scholarly journals Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit, Requires Complex Formation of β4 and HD1/Plectin, and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

1998 ◽  
Vol 142 (1) ◽  
pp. 271-284 ◽  
Author(s):  
Roel Q.J. Schaapveld ◽  
Luca Borradori ◽  
Dirk Geerts ◽  
Manuel R. van Leusden ◽  
Ingrid Kuikman ◽  
...  
Keyword(s):  
Bullous Pemphigoid ◽  
Direct Interaction ◽  
Cytoplasmic Domain ◽  
Integrin Subunit ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Fibronectin Type Iii ◽  
Activation Motif ◽  
Β4 Integrin ◽  
Two Hybrid

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.

scholarly journals Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpol with VPg and with Genetic Variants of 3AB

1998 ◽  
Vol 72 (8) ◽  
pp. 6732-6741 ◽  
Author(s):  
Wenkai Xiang ◽  
Andrea Cuconati ◽  
Debra Hope ◽  
Karla Kirkegaard ◽  
Eckard Wimmer
Keyword(s):  
Hybrid System ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Interaction ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Genome Replication ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

scholarly journals Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit.

1995 ◽  
Vol 131 (3) ◽  
pp. 807-816 ◽  
Author(s):  
S J Shattil ◽  
T O'Toole ◽  
M Eigenthaler ◽  
V Thon ◽  
M Williams ◽  
...  
Keyword(s):  
Umbilical Vein ◽  
Cytoplasmic Tail ◽  
Integrin Subunit ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Beta Integrin ◽  
Cytoplasmic Tails ◽  
Umbilical Vein Endothelial Cells ◽  
Integrin Beta 3 ◽  
Two Hybrid

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

scholarly journals Differential regulation of signaling pathways for insulin and insulin-like growth factor I.

1999 ◽  
Vol 46 (1) ◽  
pp. 51-60 ◽  
Author(s):  
W Lopaczynski
Keyword(s):  
Growth Factor ◽  
Signaling Pathways ◽  
Hybrid System ◽  
Cytoplasmic Domain ◽  
Receptor Expression ◽  
Insulin Like Growth Factor ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Igf I ◽  
Two Hybrid

The insulin receptor (IR) and the insulin-like growth factor receptor I (IGF-IR) have different functions in cell growth, apoptosis, differentation, and transformation. Although some of these differences may be explained by the relative level of receptor expression and receptor structure (alpha and beta subunits), they may also be attributed to differences in intracellular signals generated by insulin and IGF-I. The presence of hybrid receptors (IR alphabeta subunits and IGF-IR alphabeta subunits) making up the heterotetramers has added a new dimension to our understanding of the functional roles of these receptors. However, to date the results of efforts to understand the differences between these two closely related receptors have indicated mostly similarities. For example, both receptors utilize IRS-1/IRS-2 and Shc as immediate downstream adaptors, leading to activation of the Ras, Raf, ERK kinases and PI-3 kinase pathways. We have used the yeast two hybrid system to identify proteins which bind to the activated IGF-IR but not to the IR. The cytoplasmic domain of the IGF-IR was used to screen a human fetal brain library and two isoforms of the 14-3-3 family were identified. 14-3-3 proteins are a highly conserved family of proteins which have recently been shown to interact with other components of the mitogenic and apoptotic signaling pathways, including Raf, BAD, Bcr/Bcr-Abl, middle-T antigen, Ksr, PKC, PI-3 kinase, ASK1 kinase, and cdc25C phosphatase. We also identified human Grb10, an adaptor protein with SH2 domain associated with the IGF-IR beta subunit. Smith's laboratory showed that Grb10 preferentially binds to the IR in intact cells. Using the interaction trap screen (active cytoplasmic domain of the IGF-IR) 55PIK and SOCS-2 proteins were also identified. However, 55PIK and SOCS-2 also interact with the IR in the yeast two hybrid system. These studies raise the possibility that 14-3-3 and Grb10 may play a role in insulin and IGF-I signal transduction and may underlie the observed differences.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

2021 ◽  
Vol 9 (2) ◽  
pp. 304
Author(s):  
Yao Chi ◽  
Li-Long Pan ◽  
Shu-Sheng Liu ◽  
Shahid Mansoor ◽  
Xiao-Wei Wang
Keyword(s):  
Coat Protein ◽  
Molecular Mechanisms ◽  
Cotton Leaf ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Vacuolar Protein ◽  
Leaf Curl Disease ◽  
Two Hybrid ◽  
Asia Ii 1 ◽  
Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

Identification of MAL2, a Novel Member of the MAL Proteolipid Family, Though Interactions with TPD52-like Proteins in the Yeast Two-Hybrid System

Genomics ◽  
2001 ◽  
Vol 76 (1-3) ◽  
pp. 81-88 ◽  
Author(s):  
Sarah H.D Wilson ◽  
Angela M Bailey ◽  
Craig R Nourse ◽  
Marie-Geneviève Mattei ◽  
Jennifer A Byrne
Keyword(s):  
Hybrid System ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid
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