Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

beta-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of beta-arrestins in human embryonic kidney cells rescued the sequestration of beta 2-adrenergic receptor (beta 2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of beta-adrenergic receptor kinase 1. Wild-type beta 2AR sequestration was inhibited by the overexpression of two beta-arrestin mutants. These findings suggest that beta-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.

The ␤ 2 -adrenergic receptor (␤ 2 AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a ␤ 2 AR mutant (␤ 2 AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type ␤ 2 AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the ␤ 2 AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient ␤ 2 AR-Y326A mutants, demonstrated that the ␤ 2 AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the ␤-adrenergic receptor kinase 1 (␤ARK1). However, overexpression of ␤ARK1 led to the agonist-dependent phosphorylation of the ␤ 2 AR-Y326A mutant and rescue of its sequestration. ␤ARK1-mediated rescue of ␤ 2 AR-Y326A sequestration could be prevented by mutating putative ␤ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type ␤ 2 AR could be attenuated by overexpressing a dominant-negative mutant of ␤ARK1 (C 20 ␤ARK1-K220M). These findings implicate a role for ␤ARK1-mediated phosphorylation in facilitating wild-type ␤ 2 AR sequestration.The exposure of the ␤ 2 -adrenergic receptor (␤ 2 AR) 1 to catecholamines initiates its biological response via coupling to the stimulatory G protein (G s ), which then mediates the stimulation of adenylyl cyclase (1, 2). However, this receptor-mediated adenylyl cyclase response to agonist is followed by a rapid uncoupling of the receptor from its effector system, termed desensitization. The mechanisms of desensitization have been particularly well studied using the ␤ 2 AR as a model system (2). Several studies have demonstrated that the functional uncoupling of the ␤ 2 AR from G s is the consequence of its phosphorylation by one of two types of kinases (3-9). Desensitization of agonist-occupied or activated ␤ 2 AR involves phosphorylation by a growing family of G protein-coupled receptor kinases, of which ␤-adrenergic receptor kinase (␤ARK1) is a member (3,5,9). This phosphorylation serves to promote the binding of ␤-arrestin to the receptor, which when bound further uncouples the receptor (10 -12). Moreover, cAMP-dependent protein kinase (PKA) phosphorylation can desensitize the ␤ 2 AR in response to elevated intracellular cAMP levels (6 -9).In addition to functional uncoupling of the ␤ 2 AR and G s , agonist-mediated receptor internalization (sequestration) results in spatial uncoupling, such that in response to agonist plasma membrane receptors are removed to an intracellular compartment, probably into endosomes...