Amphioxus expresses both vertebrate-type and invertebrate-type dopamine D(1) receptors.

The cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates, which makes it an ideal organism for studying the molecular basis of the evolutionary transition from invertebrates to vertebrates. The biogenic amine, dopamine regulates many aspects of motor control in both vertebrates and invertebrates, and in both cases, its receptors can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. We have recently characterized one of these receptors, AmphiD1/ß, which was found to have high levels of sequence similarity to both vertebrate D1 receptors and to ß-adrenergic receptors, but functionally appeared to be a vertebrate-type dopamine D(1) receptor. Here, we report on the cloning of two further dopamine D(1) receptors (AmphiAmR1 and AmphiAmR2) from adult amphioxus cDNA libraries and their pharmacological characterisation subsequent to their expression in cell lines. AmphiAmR1 shows closer structural similarities to vertebrate D(1)-like receptors but shows some pharmacological similarities to invertebrate "DOP1" dopamine D(1)-like receptors. In contrast, AmphiAmR2 shows closer structural and pharmacological similarities to invertebrate "INDR"-like dopamine D(1)-like receptors.

Autophagosome formation in mammalian cells.

Autophagy is a fundamental intracellular trafficking pathway conserved from yeast to mammals. It is generally thought to play a pro-survival role, and it can be up regulated in response to both external and intracellular factors, including amino acid starvation, growth factor withdrawal, low cellular energy levels, endoplasmic reticulum (ER) stress, hypoxia, oxidative stress, pathogen infection, and organelle damage. During autophagy initiation a portion of the cytosol is surrounded by a flat membrane sheet known as the isolation membrane or phagophore. The isolation membrane then elongates and seals itself to form an autophagosome. The autophagosome fuses with normal endocytic traffic to mature into a late autophagosome, before fusing with lysosomes. The molecular machinery that enables formation of an autophagosome in response to the various autophagy stimuli is almost completely identified in yeast and-thanks to the observed conservation-is also being rapidly elucidated in higher eukaryotes including mammals. What are less clear and currently under intense investigation are the mechanism by which these various autophagy components co-ordinate in order to generate autophagosomes. In this review, we will discuss briefly the fundamental importance of autophagy in various pathophysiological states and we will then review in detail the various players in early autophagy. Our main thesis will be that a conserved group of heteromeric protein complexes and a relatively simple signalling lipid are responsible for the formation of autophagosomes in mammalian cells.

Regulation of autophagy by phosphatidylinositol 3-phosphate.

The simple phosphoinositide phosphatidylinositol 3-phosphate (PI(3)P) has been known to have important functions in endocytic and phagocytic traffic, and to be required for the autophagic pathway. In all of these settings, PI(3)P appears to create platforms that serve to recruit specific effectors for membrane trafficking events. In autophagy, PI(3)P may form the platform for autophagosome biogenesis.

Identification and characterization of a novel amphioxus dopamine D-like receptor.

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D(1) and D(2)) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus (Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D(1)-like receptor sequences. One of these receptors, AmphiD(1)/beta, was found to have high levels of sequence similarity to both vertebrate D(1) receptors and to beta-adrenergic receptors. Here, we report on the cloning of AmphiD(1)/beta from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD(1)/beta has a similar pharmacology to vertebrate D(1) receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits 'agonist-specific coupling' depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD(1)/beta may be an amphioxus orthologue of vertebrate beta-adrenergic receptors.

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (Branchiostoma floridae): identification, sequence analysis and phylogenetic relationship.

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.