Cell surface delivery and structural re-organization by pharmacological chaperones of an oligomerization-defective α1b-adrenoceptor mutant demonstrates membrane targeting of GPCR oligomers

Many G-protein-coupled receptors, including the α1b-adrenoceptor, form homo-dimers or oligomers. Mutation of hydrophobic residues in transmembrane domains I and IV alters the organization of the α1b-adrenoceptor oligomer, with transmembrane domain IV playing a critical role. These mutations also result in endoplasmic reticulum trapping of the receptor. Following stable expression of this α1b-adrenoceptor mutant, cell surface delivery, receptor function and structural organization were recovered by treatment with a range of α1b-adrenoceptor antagonists that acted at the level of the endoplasmic reticulum. This was accompanied by maturation of the mutant receptor to a terminally N-glycosylated form, and only this mature form was trafficked to the cell surface. Co-expression of the mutant receptor with an otherwise wild-type form of the α1b-adrenoceptor that is unable to bind ligands resulted in this wild-type variant also being retained in the endoplasmic reticulum. Ligand-induced cell surface delivery of the mutant α1b-adrenoceptor now allowed co-recovery to the plasma membrane of the ligand-binding-deficient mutant. These results demonstrate that interactions between α1b-adrenoceptor monomers occur at an early stage in protein synthesis, that ligands of the α1b-adrenoceptor can act as pharmacological chaperones to allow a structurally compromised form of the receptor to pass cellular quality control, that the mutated receptor is not inherently deficient in function and that an oligomeric assembly of ligand-binding-competent and -incompetent forms of the α1b-adrenoceptor can be trafficked to the cell surface by binding of a ligand to only one component of the receptor oligomer

The Human Gonadotropin Releasing Hormone Type I Receptor Is a Functional Intracellular GPCR Expressed on the Nuclear Membrane

The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor

Entanglement enhancement for two spins assisted by two phase kicks

Background.Pseudomonas aeruginosa is a common cause of nosocomial infections, particularly in intensive care units (ICUs). The aim of this study was to characterize P. aeruginosa clinical isolates by comparing antimicrobial susceptibility patterns with the presence of plasmids and to establish the clonal relatedness by pulsed-field gel electrophoresis (PFGE) typing. Methods. The patients included those with isolation of P. aeruginosa hospitalized for more than 48 h in the ICU from April to May 1998. Environmental and staff cultures were obtained simultaneously. Minimal inhibitory concentrations, plasmid DNA profiles, and PFGE genomic patterns of enzyme restriction chromosomal DNA were compared. Results. Sixty P. aeruginosa isolates were obtained from 197 clinical specimens, 178 environmental samples, and 47 hand cultures of personnel. Antimicrobial resistance was as follows: tobramycin 100%; ticarcillin, cefotaxime, ceftriaxone, ceftazidime, and gentamicin 80%; cefepime 60%; amikacin, ticarcillin/clavulanate, imipenem, and meropenem 40%; piperacillin and norfloxacin 20%; carbenicillin 12%, and ciprofloxacin 0%. Plasmids were detected in 11 isolates (18%). PFGE typing showed that 23 isolates belonged to a common clone (pattern A), identified from five patients, two nurses, and 10 environmental samples. Ten isolates were grouped in four clusters and 27 isolates had unrelated genomic patterns. There was no relationship among DNA genomic patterns, plasmid profiles, and susceptibility patterns. Conclusions. PFGE demonstrated the existence of a common clone in a critical care area. Reinforcement of infection control measures is needed to avoid horizontal transmission and severe infections. Copyright " 2001 IMSS.",,,,,,"10.1016/S0188-4409(01)00267-3",,,"http://hdl.handle.net/20.500.12104/41261","http://www.scopus.com/inward/record.url?eid=2-s2.0-0034981240&partnerID=40&md5=b1c9d05e0380724563de18282e321139",,,,,,"3",,"Archives of Medical Research",,"23

Epidemiologic study of Pseudomonas aeruginosa in critical patients and reservoirs

Background.Pseudomonas aeruginosa is a common cause of nosocomial infections, particularly in intensive care units (ICUs). The aim of this study was to characterize P. aeruginosa clinical isolates by comparing antimicrobial susceptibility patterns with the presence of plasmids and to establish the clonal relatedness by pulsed-field gel electrophoresis (PFGE) typing. Methods. The patients included those with isolation of P. aeruginosa hospitalized for more than 48 h in the ICU from April to May 1998. Environmental and staff cultures were obtained simultaneously. Minimal inhibitory concentrations, plasmid DNA profiles, and PFGE genomic patterns of enzyme restriction chromosomal DNA were compared. Results. Sixty P. aeruginosa isolates were obtained from 197 clinical specimens, 178 environmental samples, and 47 hand cultures of personnel. Antimicrobial resistance was as follows: tobramycin 100%; ticarcillin, cefotaxime, ceftriaxone, ceftazidime, and gentamicin 80%; cefepime 60%; amikacin, ticarcillin/clavulanate, imipenem, and meropenem 40%; piperacillin and norfloxacin 20%; carbenicillin 12%, and ciprofloxacin 0%. Plasmids were detected in 11 isolates (18%). PFGE typing showed that 23 isolates belonged to a common clone (pattern A), identified from five patients, two nurses, and 10 environmental samples. Ten isolates were grouped in four clusters and 27 isolates had unrelated genomic patterns. There was no relationship among DNA genomic patterns, plasmid profiles, and susceptibility patterns. Conclusions. PFGE demonstrated the existence of a common clone in a critical care area. Reinforcement of infection control measures is needed to avoid horizontal transmission and severe infections. Copyright © 2001 IMSS