J.L. Arriza

Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non-nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L-glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology.

Homologous or agonist-specific desensitization of beta 2-adrenergic receptors (beta 2AR) is mediated by the beta-adrenergic receptor kinase (beta ARK) which specifically phosphorylates the agonist-occupied form of the receptor. However, the capacity of beta ARK-phosphorylated beta 2AR to stimulate Gs in a reconstituted system is only minimally impaired. Recently, a protein termed beta-arrestin, was cloned from a bovine brain cDNA library and found to quench phosphorylated beta 2AR-coupling to Gs. Utilizing a low stringency hybridization technique to screen a rat brain cDNA library, we have now isolated cDNA clones representing two distinct beta-arrestin-like genes. One of the cDNAs is the rat homolog of bovine beta-arrestin (beta-arrestin1). In addition, we have isolated a cDNA clone encoding a novel, beta-arrestin-related protein which we have termed beta-arrestin2. Overall, beta-arrestin2 exhibits 78% amino acid identity with beta-arrestin1. The primary structure of these proteins delineates a family of proteins that regulates receptor coupling to G proteins. The capacity of purified beta-arrestin1, beta-arrestin2, and arrestin to inhibit the coupling of phosphorylated receptors to their respective G proteins were assessed in a reconstituted beta 2AR-Gs system and in a reconstituted rhodopsin-GT system. beta-Arrestin2 was equipotent to beta-arrestin1 and specifically inhibited beta 2AR function. Conversely, arrestin inhibited rhodopsin coupling to GT, whereas beta-arrestin1 and beta-arrestin2 were at least 20-fold less potent in this system. beta-Arrestin1 and beta-arrestin2 are predominantly localized in neuronal tissues and in the spleen. However, low mRNA levels can be detected in most peripheral tissues. In the central nervous system, beta-arrestin2 appears to be even more abundant than beta-arrestin1. Immunohistochemical analysis of the tissue distribution of beta-arrestin1 and beta-arrestin2 in rat brain shows extensive, but heterogenous, neuronal labeling of the two proteins. They are found in several neuronal pathways suggesting that they have relatively broad receptor specificity regulating many G protein-coupled receptors. Furthermore, immunoelectron microscopy shows that the beta-arrestins are appropriately situated at postsynaptic sites to act in concert with beta ARK to regulate G protein-coupled neurotransmitter receptors.

A cDNA was isolated from human brain that encodes an amino acid sequence 34-39% identical to previously published glutamate transporter sequences. Injection of RNA transcribed from this cDNA into Xenopus oocytes resulted in expression of a transport activity with the properties of the neutral amino acid uptake system ASC. Superfusion of alanine, serine, and cysteine evoked sodium-dependent inward currents in voltage-clamped oocytes expressing the transporter. These currents were dose-dependent, stereospecific, and saturable, with Km values ranging from 29 to 88 microM. Northern blot analyses revealed ubiquitous expression of this gene, termed ASCT1, consistent with the general metabolic role ascribed to system ASC.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.