AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34 ؉ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5-untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissuespecific stem and progenitor cells. (Blood.

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.

A wide range of extracellular signals are transduced by G protein-coupled receptors (GPCRs). When activated by ligands, GPCRs can activate associated heterotrimeric guanine nucleotide-binding proteins (G proteins), which in turn act on various effectors. Increasing evidence indicates that GPCRs also signal independently of heterotrimeric G proteins. Several GPCRs directly interact with Src-family kinases. Here, we discuss the evidence for direct interaction and activation of Src-family kinases by GPCRs and data that suggest that agonist dosage provides a mechanism by which GPCRs can switch between G protein-dependent and G protein-independent signaling.

ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+ o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+ o, consistent with coupling between the responses to pHi and K+ o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4 + >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+ o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+ o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+ o by altering the properties of the selectivity filter.

Background: The C-terminal Src kinase (Csk) is known as a tumor suppressor, but Src-independent function is unclear. Results: eEF2 is a new protein substrate of Csk. Conclusion: eEF2 phosphorylation and SUMOylation promote its proteolytic cleavage and nuclear localization. Significance: Our findings suggest that a tyrosine kinase can be both a tumor suppressor and a promoter through regulation of different substrate proteins.