Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

BACKGROUND: Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta (2)-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta(2)-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase. RESULTS: The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta(2)-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta(2)-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β(2)-adrenergic receptor for those of Ste2 creates a Ste2/beta(2)-adrenergic receptor chimera displaying insulin-stimulated internalization. CONCLUSION: Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins

Genomic features of the human dopamine transporter gene and its potential epigenetic States: implications for phenotypic diversity.

Human dopamine transporter gene (DAT1 or SLC6A3) has been associated with various brain-related diseases and behavioral traits and, as such, has been investigated intensely in experimental- and clinical-settings. However, the abundance of research data has not clarified the biological mechanism of DAT regulation; similarly, studies of DAT genotype-phenotype associations yielded inconsistent results. Hence, our understanding of the control of the DAT protein product is incomplete; having this knowledge is critical, since DAT plays the major role in the brain's dopaminergic circuitry. Accordingly, we reevaluated the genomic attributes of the SLC6A3 gene that might confer sensitivity to regulation, hypothesizing that its unique genomic characteristics might facilitate highly dynamic, region-specific DAT expression, so enabling multiple regulatory modes. Our comprehensive bioinformatic analyzes revealed very distinctive genomic characteristics of the SLC6A3, including high inter-individual variability of its sequence (897 SNPs, about 90 repeats and several CNVs spell out all abbreviations in abstract) and pronounced sensitivity to regulation by epigenetic mechanisms, as evident from the GC-bias composition (0.55) of the SLC6A3, and numerous intragenic CpG islands (27 CGIs). We propose that this unique combination of the genomic features and the regulatory attributes enables the differential expression of the DAT1 gene and fulfills seemingly contradictory demands to its regulation; that is, robustness of region-specific expression and functional dynamics

Genotype and Ancestry Modulate Brain's DAT Availability in Healthy Humans

The dopamine transporter (DAT) is a principal regulator of dopaminergic neurotransmission and its gene (the SLC6A3) is a strong biological candidate gene for various behavioral- and neurological disorders. Intense investigation of the link between the SLC6A3 polymorphisms and behavioral phenotypes yielded inconsistent and even contradictory results. Reliance on objective brain phenotype measures, for example, those afforded by brain imaging, might critically improve detection of DAT genotype-phenotype association. Here, we tested the relationship between the DAT brain availability and the SLC6A3 genotypes using an aggregate sample of 95 healthy participants of several imaging studies. These studies employed positron emission tomography (PET) with [11C]cocaine wherein the DAT availability was estimated as Bmax/Kd; while the genotype values were obtained on two repeat polymorphisms - 3-UTR- and intron 8- VNTRs

Regression coefficients of DAT availability and age in genotype groups.

<p><b><u>Abbreviations:</u></b><i>b</i>-(beta weights), standardized coefficient; <i>p</i>-significance, values<0.05 are marked with*; <i>t</i>-score (t-statistic is the coefficient divided by its standard error), significant at ItI>2.</p

Increased recombination rate in the VNTR-encompassing regions of the <i>SLC6A3</i> locus.

<p>Both VNTR regions are within the recombination Hotspots indicated by red arrows (HapMap Data Release 27 PhaseII+III, Feb09, on NCBI B36 assembly). The green slashed box encloses a recombination hot spot (upper panel) that is projected to the intron8 (green arrow, bottom panel); the blue dotted box encloses a recombination hot spot (upper panel) mapped to the 3′UTR – region (green arrow).</p

Haplotype structure of the <i>SLC6A3</i> 3′UTR locus in African-American- and Caucasian- populations.

<p>Graphical representation of the haplotype structure of the 3′-UTR region: African-American population - Panel A, top; Caucasian population - Panel B, top. Color scheme: blue – homozygotes, common allele, yellow – homozygotes, rare allele, red – heterozygotes, and grey – undetermined. Data are retrieved using Genome Variation Server (<a href="http://gvs.gs.washington.edu/GVS" target="_blank">http://gvs.gs.washington.edu/GVS</a>). Bottom of the panels illustrates corresponding haplotype maps: Confidence Bounds Color scheme: Strong evidence of LD – dark grey, uninformative- light grey and white color indicates strong evidence of recombination. More of dark grey in CE (red arrow) is indicative of the presence of LD block which is population-specific, whereas the light grey and the white color are predominant in the haplotype map of AA population (green arrow), indicate high recombination rate.</p

Sample Demographics.

<p>Ethnic group “Others” included 5 Hispanics- and 5 individuals of more than one race.</p