Ligand-independent and EGF receptor-supported transactivation: lessons from beta2-adrenergic receptor signalling

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) is currently understood to be mediated by matrix metalloproteases (MMPs) and the release of EGF-like ligands. This ligand-mediated process also suggests that downstream of EGFR the signalling in response to GPCR ligands or EGF appears to be indistinguishable. Here we provide evidence that transactivation of EGFR by the beta2-adrenergic receptor (beta2-AR) is independent of MMPs and results in an incomplete downstream signalling involving extracellular signal-activated kinase (ERK) but not PLCgamma1 and Akt. In contrast, beta2-AR has the ability to activate PLCgamma1 when the EGFR is primed either by co-stimulation with EGF or by increased basal activity due to over-expression. In that way but not via the beta2-AR-mediated transactivation the EGFR docking sites pY992 and pY1173 may be generated which are critical for PLCgamma1. This EGFR-supported transactivation is strongly dependent on EGFR tyrosine kinase, c-Src, and the c-Src-specific EGFR tyrosine residue 845 and represents a novel paradigm of EGFR transactivation.     

Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias)

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.     

Analysis of the tellurite resistance determinant on the pNT3B derivative of the pTE53 plasmid from uropathogenic Escherichia coli

We have found and sequenced a significant part of the previously described tellurite resistance determinant on mini-Mu derivative pPR46, named pNT3B, originally cloned from a large conjugative plasmid pTE53, found in Escherichia coli. This plasmid contains genes essential for tellurite resistance, together with the protective region bearing genes terX, Y, W, and the conserved spacing region bearing several ORFs of unknown function. Computer analysis of obtained sequence revealed a close similarity to the formerly described ter operons found on the Serratia marcescens plasmid R478 and the chromosome of Escherichia coli O157:H7. This finding confirms the presence of a whole region on the large conjugative plasmid that pTE53 originated from a uropathogenic E. coli strain, and suggests its possible role in horizontal gene transfer, resulting in the development of new pathogenic E. coli strains.     

Masking of Transmembrane‐Based Retention Signals Controls ER Export of γ‐Secretase

γ‐Secretase is critically involved in the Notch pathway and in Alzheimer's disease. The four subunits of γ‐secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER‐retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of γ‐secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER‐retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER‐retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent γ‐secretase complex could mask TMD‐based ER‐retention/retrieval signals to allow plasma membrane transport of fully assembled γ‐secretase.     

Spectral properties of Trp182, Trp194, and Trp250 on the alpha subunit of bacterial luciferase.

The phosphorescence and fluorescence properties of bacterial luciferase (alphabeta) mutants from Xenorhabdus luminescens were investigated. All tryptophans in the alpha and beta subunits were replaced with tyrosines except for one or two tryptophans in the alpha subunit. Because one luciferase mutant (W250) retained only a single tryptophan in the alpha subunit while two other mutants (W182/250 and W194/250) each contained two tryptophans in the alpha subunit, it was possible to deduce the spectral properties of these specific tryptophans (Trp182, Trp194, Trp250). Analyses of the phosphorescence properties were particularly revealing as only a single phosphorescence emission peak at 411-414 nm was observed for the W250 and W194/250 mutants while peaks at 409 and 414 nm could be clearly observed for the W182/250 mutant. Coupled with intrinsic fluorescence quenching experiments, these results show that alphaTrp182 is in a distinctly polar environment while alphaTrp250 is in a hydrophobic region and illustrate the advantages of using phosphorescence to recognize different microenvironments for tryptophan residues.     

prbA,a gene coding for an esterase hydrolyzing parabens in enterobacter cloacae and Enterobacter gergoviae strains

The new gene prbA encodes an esterase responsible for the hydrolysis of the ester bond of parabens in Enterobacter cloacae strain EM. This gene is located on the chromosome of strain EM and was cloned by several PCR approaches. The prbA gene codes for an immature protein of 533 amino acids, the first 31 of which represent a proposed signal peptide yielding a mature protein of a putative molecular mass of 54.6 kDa. This enzyme presents analogies with other type B carboxylesterases, mainly of eukaryotic origin. The cloning and expression of the prbA gene in a strain of Escherichia coli previously unable to hydrolyze parabens resulted in the acquisition of a hydrolytic capacity comparable to the original activity of strain EM, along with an increased resistance of the transformed strain to methyl paraben. The presence of homologues of prbA was tested in additional ubiquitous bacteria, which may be causative factors in opportunistic infections, including Enterobacter gergoviae, Enterobacter aerogenes, Pseudomonas agglomerans, E. coli, Pseudomonas aeruginosa, and Burkholderia cepacia. Among the 41 total strains tested, 2 strains of E. gergoviae and 1 strain of Burkholderia cepacia were able to degrade almost completely 800 mg of methyl paraben liter(-1). Two strains of E. gergoviae, named G1 and G12, contained a gene that showed high homology to the prbA gene of E. cloacae and demonstrated comparable paraben esterase activities. The significant geographical distance between the locations of the isolated E. cloacae and E. gergoviae strains suggests the possibility of an efficient transfer mechanism of the prbA gene, conferring additional resistance to parabens in ubiquitous bacteria that represent a common source of opportunistic infections.     

Muscarinic M2 receptors mediate transactivation of EGF receptor through Fyn kinase and without matrix metalloproteases

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) has been attributed to the activation of matrix metalloproteases (MMPs) and the release of EGF family ligands such as HB-EGF. This mode of transactivation leads to signalling downstream of EGFR which is indistinguishable from that induced by the ligand. Here we provide evidence that in the COS-7 cell model EGFR transactivation via the muscarinic M2 receptor (M2R) is independent of MMPs and results in an incomplete EGFR signalling including ERK and Akt but not PLCgamma1. Using dominant-negative mutants of c-Src and Fyn and Src-deficient SYF cells as well as by co-immunoprecipitation studies, we can demonstrate that the M2R-mediated transactivation of EGFR specifically involves Fyn but not c-Src or Yes. This specific role of Fyn can be verified in SH-SY5Y human neuroblastoma cells with endogenously expressed M2 receptors.