beta-arrestin1 interacts with the catalytic domain of the tyrosine kinase c-SRC. Role of beta-arrestin1-dependent targeting of c-SRC in receptor endocytosis

beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2-adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta-arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.     

Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.     

Bypassing the kinetic trap of serpin protein folding by loop extension

The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring-loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of alpha1-antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of alpha1-antitrypsin converts the molecule into a more stable state. Remarkably, a 30-residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major beta-sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of alpha1-antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.     

A Physical Interaction of UvrD with Nucleotide Excision Repair Protein UvrB

The dual-incision nature of the reaction of UV-irradiated DNA catalyzed by the UvrABC complex potentially leads to excision of a damaged fragment. However, neither fragment release under nondenaturing conditions nor the UvrBC proteins are turned over. The addition of the UvrD protein to the incised DNA-UvrBC complex results in excision of the incised damaged strand and in the turnover of the UvrC protein. In an effort to better understand the involvement of UvrD in the excision step, immunoprecipitation was used to detect interacting proteins with UvrD in the DNA repair. In this communication, it is shown that UvrA and UvrB are precipitated with UvrD in solution but the UvrAB complex is not. In the incision complex, UvrB could be precipitated and the preincubation of UvrD with UvrB revealed an inhibitory effect on the turnover of the incision complex. These data imply that UvrB in the incision complex seems to recruit UvrD to the 3 incised site of the incised strand by protein-protein interaction and to allow initiation of unwinding by UvrD from the resulting nick in a 3 to 5 direction.     

Novel mutations of the PKD1 gene in Korean patients with autosomal dominant polycystic kidney disease

The gene for the most common form of autosomal dominant polycystic kidney disease (ADPKD), PKD1, has recently been characterized and shown to encode an integral membrane protein, polycystin-1, which is involved in cell-cell and cell-matrix interactions. Until now, approximately 30 mutations of the 3' single copy region of the PKD1 gene have been reported in European and American populations. However, there is no report of mutations in Asian populations. Using the polymerase chain reaction and single-strand conformation polymorphism (SSCP) analysis, 91 Korean patients with ADPKD were screened for mutation in the 3' single copy region of the PKD1 gene. As a result, we have identified and characterized six mutations: three frameshift mutations (11548del8bp, 11674insG and 12722delT), a nonsense mutation (Q4010X), and two missense mutations (R3752W and D3814N). Five mutations except for Q4010X are reported here for the first time. Our findings also indicate that many different mutations are likely to be responsible for ADPKD in the Korean population. The detection of additional disease-causing PKD1 mutations will help in identifying the location of the important functional regions of polycystin-1 and help us to better understand the pathophysiology of ADPKD.     

Importance of phosphatidylethanolamine for the interaction of apocytochrome c with model membranes containing phosphatidylserine

The effect of phosphatidylethanolamine (PE) on the binding of apocytochrome c to model membranes was examined. When 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of the standard vesicles composed of 80% of this lipid and 20% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) was gradually replaced with upward of 50% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), the binding increased appreciably. Ca(2+), causing the phase separation of PS, also brought about increased binding of apocytochrome c in the PC/PS system, underlining the importance of PS properties in membranes for the protein binding. The resonance energy transfer between Trp-59 in apocytochrome c and pyrene-PS incorporated into bilayers showed that the replacement of PC with PE increased the extent of apocytochrome c penetration into membranes by a PE concentration-dependent manner. However, in the absence of PS, PE had no apparent effect on these functions of apocytochrome c, suggesting that PE-induced change(s) of acidic membrane properties is important to the association of apocytochrome c with vesicles. From the observations that the excimer to monomer fluorescence ratio of pyrene-PS increased and the fluorescence of NBD-PS was quenched with increasing concentration of PE, it was deduced that PE caused PS-enriched domains in PC/PE/PS membranes. The colocalization of pyrene-PS with BODIPY-PS by PE further supported the possibility. We suggest that PE-induced formation of PS-enriched domains acts as binding sites for apocytochrome c in membranes.     

Identification of trait-improving quantitative trait loci alleles from a wild rice relative,Oryza rufipogon.

Wild species are valued as a unique source of genetic variation, but they have rarely been used for the genetic improvement of quantitative traits. To identify trait-improving quantitative trait loci (QTL) alleles from exotic species, an accession of Oryza rufipogon, a relative of cultivated rice, was chosen on the basis of a genetic diversity study. An interspecific BC2 testcross population (V20A/O. rufipogon//V20B///V20B////Ce64) consisting of 300 families was evaluated for 12 agronomically important quantitative traits. The O. rufipogon accession was phenotypically inferior for all 12 traits. However, transgressive segregants that outperformed the original elite hybrid variety, V20A/Ce64, were observed for all traits examined. A set of 122 RFLP and microsatellite markers was used to identify QTL. A total of 68 significant QTL were identified, and of these, 35 (51%) had beneficial alleles derived from the phenotypically inferior O. rufipogon parent. Nineteen (54%) of these beneficial QTL alleles were free of deleterious effects on other characters. O. rufipogon alleles at two QTL on chromosomes 1 and 2 were associated with an 18 and 17% increase in grain yield per plant, respectively, without delaying maturity or increasing plant height. This discovery suggests that the innovative use of molecular maps and markers can alter the way geneticists utilize wild and exotic germplasm.