Cloning and sequencing techniques now allow us to characterize genes directly instead of having to deduce their properties from their effects. This new genetics reaches its apotheosis in the plan to obtain the complete DNA sequence of the human genome, but this is far beyond the capacity of present sequencing methods. Small 'model' genomes, 'such as those of Escherichia coli (4.7 megabases (Mb) and yeast (14 Mb), or even those of Caenorhabditis elegans (100 Mb) and Drosophila (165 Mb), are better scaled to existing technology. The yeast genome will contain genes with functions common to all eukaryotic cells, and those of simple multicellular organisms may throw light on the genetic specification of more complex functions. However, vertebrates differ in their morphology and development, so the ideal model would be a vertebrate genome of minimum size and complexity but with maximum homology to the human genome. Here we report the characterization of the small genome (400 Mb) of the tetraodontoid fish, Fugu rubripes. A random sequencing approach supported by gene probing shows that the haploid genome contains 400 Mb of DNA, of which more that 90% is unique. This genome is 7.5 times smaller than the human genome and because it has a similar gene repertoire it is the best model genome for the discovery of human genes.

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7). Here we show that GRK6-deficient mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine. In addition, these mice demonstrate an enhanced coupling of striatal D2-like dopamine receptors to G proteins and augmented locomotor response to direct dopamine agonists both in intact and in dopamine-depleted animals. The present study indicates that postsynaptic D2-like dopamine receptors are physiological targets for GRK6 and suggests that this regulatory mechanism contributes to central dopaminergic supersensitivity.

The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA.The four GRK4 proteins have been expressed, and all incorporate [ 3 H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/ chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified ␤ 2 -adrenergic receptor, indicating that GRK4 is a functional protein kinase.

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

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The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.