The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.

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.

Members of the G protein-coupled receptor kinase family that phosphorylate the beta2-adrenergic receptor facilitate sequestration.

We recently reported that a beta2-adrenergic receptor (beta2AR) mutant, Y326A, defective in its ability to sequester in response to agonist stimulation was a poor substrate for G protein-coupled receptor kinase (GRK)-mediated phosphorylation; however, its ability to be phosphorylated and sequestered could be restored by overexpressing GRK2 [Ferguson et al. (1995) J. Biol. Chem. 270, 24782]. In the present report, we tested the ability of each of the known GRKs (GRK1-6) to phosphorylate and rescue the sequestration of the Y326A mutant in HEK-293 cells. We demonstrate that in addition to GRK2, GRK3-6 can phosphorylate the Y326A mutant and rescue its sequestration; however, GRK1 was totally ineffective in rescuing either the phosphorylation or the sequestration of the mutant receptor. We found that the agonist-dependent rescue of Y326A mutant phosphorylation by GRK2, -3, and -5 was associated with the agonist-dependent rescue of sequestration. In contrast, overexpression of GRK4 and -6 led mainly to agonist-independent phosphorylation of the Y326A mutant accompanied by increased basal receptor sequestration. Our results demonstrate that phosphorylation per se, but not the interaction with a specific GRK, is required to facilitate beta2AR sequestration.

Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization.

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 conserved seven-transmembrane sequence NP(X)2,3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor.

The beta 2-adrenergic receptor (beta 2AR) is a member of a large superfamily of seven transmembrane domain, G-protein-coupled receptors. Within the putative seventh transmembrane domain of the beta 2AR is a sequence of amino acids, NPLIY, which is conserved with minor variations in all members of the superfamily. Previously it was demonstrated that mutation of tyrosine residue 326 to an alanine abolished agonist promoted sequestration of this mutant without affecting its ability to maximally stimulate adenylyl cyclase in membranes [Barak, L.S., Tiberi, M., Freedman, N.J., Kwatra, M.M., Lefkowitz, R.J., & Caron M.J. (1994) J Biol. Chem. 269, 2790-2795]. In the present study we characterized the NPLIY amino acid sequence in an attempt to determine how it can affect the agonist-mediated sequestration of the beta 2AR and to test whether it is a functional motif. We find that point mutations of the most conserved amino acids, N, P, and Y, in this sequence affect several other receptor properties in addition to sequestration. Mutation of asparagine 322 to an alanine resulted in complete uncoupling of the receptor, loss of high-affinity agonist binding, and abolition of receptor sequestration, down-regulation, and phosphorylation. In contrast, a conservative mutation of this residue to an aspartic acid (as found in the thrombin receptor) resulted in an improvement of G-protein coupling without adversely affecting other receptor properties. Substitution of proline residue 323 with an alanine residue resulted in a receptor with mild deficits in sequestration and coupling, a reduced agonist-mediated phosphorylation, and no change in down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.

The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. 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 beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.

The human D1A dopamine receptor gene promoter directs expression of a reporter gene to the central nervous system in transgenic mice.

Dopamine receptors are involved in many aspects of dopaminergic neurotransmission including regulation of motor control, cognition, affect and neuroendocrine function. The D1A receptor is the most widely distributed dopamine receptor in the brain and is expressed at high levels in the striatum and nucleus accumbens, but is also found throughout cortical, limbic, hypothalamic and thalamic brain regions. We have cloned a 6.4 kb fragment 5' of the human D1A dopamine receptor gene and shown that this region activates transcription of the chloramphenicol acetyltransferase (CAT) gene in a cell-specific manner. To study the expression of these sequences in vivo we analyzed the expression of the E. coli lac Z gene under the regulation of the 6.4 kb fragment in transgenic mice. Expression of the transgene was primarily detected in the brain, with only low levels detected in peripheral tissues. The 5' flanking sequences were able to direct the tissue-specific expression of lac Z in three different lines of transgenic mice, to a number of brain regions including the caudate-putamen, thalamus, amygdala, cerebral cortex, hippocampus and hypothalamus. Greatest expression of the lac Z gene was detected in areas of the thalamus and amygdaloid complex. In the striatum, beta-galactosidase activity was restricted to neurons within the matrix and was not detected within striosomes. Results of this study demonstrate that the 6.4 kb region upstream of the human D1A receptor gene is sufficient to confer tissue-specific expression in the CNS of transgenic mice. Furthermore, expression of the transgene to neurons within the matrix of the striatum, but not the striosomes suggests that expression of the D1A receptor may be regulated differently within these areas.

Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels.

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.