The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.     

Microarray analysis of bacterial gene expression: towards the regulome

Microarray technology allows co-regulated genes to be identified. In order to identify genes that are controlled by specific regulators, gene expression can be compared in mutant and wild-type bacteria. However, there are a number of pitfalls with this approach; in particular, the regulator may not be active under the conditions in which the wild-type strain is cultured. Once co-regulated genes have been identified, proteinbinding motifs can be identified. By combining these data with a map of promoters, or operons (the operome), the regulatory networks in the cell (the regulome) can start to be built up.     

Apoaequorin monitors degradation of endoplasmic reticulum (ER) proteins initiated by loss of ER Ca(2+)

Apoaequorin was targeted to the cytosol, nucleus, and endoplasmic reticulum of HeLa cells in order to determine the effect of Ca(2+) release from the ER on protein degradation. In resting cells apoaequorin had a rapid half-life (ca. 20-30 min) in the cytosol or nucleus, but was relatively stable for up to 24 h in the ER (t(1/2) > 24 h). However, release of Ca(2+) from the ER, initiated by the addition of inhibitors of the ER Ca(2+)/Mg(2+) ATPase such as 2 microM thapsigargin or 1 microM ionomycin, initiated rapid loss of apoaequorin in the ER, but had no detectable effect on apoaequorin turnover in the cytosol nor the nucleus. This loss of apoprotein was not the result of secretion into the external fluid, and could not be inhibited by inhibitors of protein degradation by proteosomes. Proteolysis of apoaequorin in cell extracts (t(1/2) < 20 min) was completely inhibited in the presence of 1 mM Ca(2+), and this effect was independent of the ER retention signal KDEL at the C-terminus. Proteolysis was unaffected by the presence of selected serine protease inhibitors, or 10 microM Zn(2+), a known caspase-3 inhibitor. The results show that apoaequorin can monitor proteolysis of ER proteins activated by loss of ER Ca(2+). Several Ca(2+)-binding proteins exist in the ER, acting as the Ca(2+) store and chaperones. Our results have important implications both for the role of ER Ca(2+) in cell activation and stress and when using aequorin for monitoring free ER Ca(2+) over long time periods.     

Genetic analysis of the exed region in mouse

The extraembryonic ectoderm development (exed) mutant phenotype was described in mice homozygous for the c(6H) deletion, a radiation-induced deletion in the tyrosinase region of mouse Chromosome 7. These mutants fail to gastrulate and die around embryonic day 8.0. Several genes including, for example, embryonic ectoderm development (eed), are deleted in the c(6H) mutants; however, the portion of the chromosome responsible for the more severe exed phenotype is localized to a 20-kb region called the "exed-critical region." To understand the genetics behind the exed phenotype, we analyzed this region in two ways. First, to determine whether the 20-kb exed-critical region alone causes the mutant phenotype, we removed it from a wild-type chromosome. The resulting mice homozygous for this deletion were viable and fertile, indicating that the 20-kb exed-critical region by itself is not sufficient to cause the phenotype when deleted. We then sequenced the 20-kb exed-critical region and no expressed exons were found. Several short matches to GenBank Expressed Sequence Tag (EST) databases were identified; however, none of these ESTs mapped to the region. Taken together, these results indicate that the exed phenotype may either be a position effect on a distal gene caused by the c(6H) breakpoint or the result of composite effects of nullizygosity of multiple genes in the deletion homozygotes.     

An adrenaline (and gold?) rush for the GPCR community.

G-Protein-coupled receptors are one of the largest protein families found in metazoans. Using several novel strategies, the first atomic resolution structures of a receptor that is activated by a diffusible ligand have been determined.     

Age validation and variation in growth,mortality and population structure of Liza argentea and Myxus elongatus (Mugilidae) in two temperate Australian estuaries

This study investigated variation in the rates of growth and mortality, and age and fork‐length (L_F) compositions of two exploited species of Mugilidae, Liza argentea and Myxus elongatus, in two south‐east Australian estuaries (Lake Macquarie and St Georges Basin). An ageing protocol was developed by counting opaque growth zones on sectioned otoliths which was validated by periodically examining the otoliths of captive‐reared young‐of‐the‐year fishes, and marginal increment analysis of wild fishes. The maximum recorded age was 17 years for L. argentea and 12 years for M. elongatus, which is greater than generally observed in other species of mugilids. Growth models of each species significantly differed between sexes and, except for male L. argentea, between estuaries. Fishes from Lake Macquarie generally had a greater mean L_F at age than those from St Georges Basin and females of both species generally attained a greater maximum L_F and age than males. Gillnet catches of L. argentea were of similar L_F and age compositions in both estuaries, whereas the age composition of catches of M. elongatus in Lake Macquarie contained a greater proportion of younger fish. Estimates of total, natural and fishing mortality were greater for M. elongatus than L. argentea across both estuaries, and estimates of total mortality were greatest for both species in Lake Macquarie. The data indicate that neither species has been overfished in these estuaries.