Identification and allele-specific silencing of the mutant huntingtin allele in Huntington's disease patient-derived fibroblasts.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by the expression of mutant huntingtin protein (Htt). Suppression of Htt expression, using RNA interference, might be an effective therapy. However, if reduction of wild-type protein is not well tolerated in the brain, it may be necessary to suppress just the product of the mutant allele. We present a small interfering RNA (siRNA) that selectively reduces the endogenous mRNA for a heterozygous HD donor's pathogenic allele by approximately 80% by specifically targeting a single-nucleotide polymorphism (SNP) located several thousand bases downstream from the disease-causing mutation. In addition, we show selective suppression of endogenous mutant Htt protein, using this siRNA. We further present a method, using just a heterozygous patient's own mRNA, to determine which SNP variants correspond to the mutant allele. The method may be useful in any disorder in which a targeted SNP is far downstream from the pathogenic mutation. These results indicate that allele-specific treatment for Huntington's disease may be clinically feasible and practical.

Adjuvant arthritis induces down-regulation of G protein-coupled receptor kinases in the immune system.

G protein-coupled receptors (GPCR) play a crucial role in the regulation of the immune response by, e.g., chemokines, PGs, and beta(2)-adrenergic agonists. The responsiveness of these GPCRs is turned off by the family of G protein-coupled receptor kinases (GRK1-6). These kinases act by phosphorylating the GPCR in an agonist-dependent manner, resulting in homologous desensitization of the receptor. Although GRKs are widely expressed throughout the body, leukocytes express relatively high levels of GRKs, in particular GRK2, -3, and -6. We investigated whether in vivo the inflammatory disease adjuvant arthritis (AA) induces changes in GRK expression and function in the immune system. In addition, we analyzed whether the systemic effects of AA also involve changes in GRKs in nonimmune organs. At the peak of the inflammatory process, we observed a profound down-regulation of GRK2, -3, and -6 in splenocytes and mesenteric lymph node cells from AA rats. Interestingly, no changes in GRK were observed in thymocytes and in nonimmune organs such as heart and pituitary. During the remission phase of AA, GRK levels in spleen and mesenteric lymph nodes are returning to baseline levels. The decrease in GRK2 at the peak of AA is restricted to CD45RA(+) B cells and CD4(+) T cells, and was not observed in CD8(+) T cells. In conclusion, we demonstrate in this study, for the first time, that an inflammatory process in vivo induces a tissue-specific down-regulation of GRKs in the immune system.

Patients with systemic lupus erythematosus differ from healthy controls in their immunological response to acute psychological stress.

Clinical observations suggest that psychological stress induces exacerbation of disease activity in patients with systemic lupus erythematosus (SLE). In order to determine whether SLE patients differ from healthy controls in their stress response, we analyzed heart rate, blood pressure, catecholamine concentration, lymphocyte subpopulations, natural killer (NK) cell activity, and expression of beta-adrenoceptors on PBMC before, immediately after, and 1 h after a public speaking task in 15 SLE patients and 15 healthy subjects. Both groups demonstrated similar psychological, cardiovascular, and neuroendocrine responses to acute stress. However, natural killer (CD16(+)/CD56(+)) cell numbers transiently increased after stress exposure, with significantly less pronounced changes in SLE patients. In addition, NK activity increased in healthy controls (n = 8) but not in SLE patients (n = 4) after acute stress. Furthermore, the number of beta(2)-adrenoceptors on PBMC significantly increased only in healthy subjects (n = 8) after stress but not in SLE patients (n = 7). These data indicate that SLE patients differ from healthy controls in stress-induced immune responses.

Decreased expression and activity of G-protein-coupled receptor kinases in peripheral blood mononuclear cells of patients with rheumatoid arthritis.

Beta2-Adrenergic and chemokine receptor antagonists delay the onset and reduce the severity of joint injury in rheumatoid arthritis. beta2-Adrenergic and chemokine receptors belong to the G-protein-coupled receptor family whose responsiveness is turned off by the G-protein-coupled receptor kinase family (GRK-1 to 6). GRKs phosphorylate receptors in an agonist-dependent manner resulting in receptor/G-protein uncoupling via subsequent binding of arrestin proteins. We assessed the activity of GRKs in lymphocytes of rheumatoid arthritis (RA) patients by rhodopsin phosphorylation. We found a significant decrease in GRK activity in RA subjects that is mirrored by a decrease in GRK-2 protein expression. Moreover, GRK-6 protein expression is reduced in RA patients whereas GRK-5 protein levels were unchanged. In search of an underlying mechanism, we demonstrated that proinflammatory cytokines induce a decrease in GRK-2 protein levels in leukocytes from healthy donors. Since proinflammatory cytokines are abundantly expressed in RA, it may provide an explanation for the decrease in GRK-2 expression and activity in patients. No changes in beta2-adrenergic receptor number and Kd were detected. However, RA patients showed a significantly increased cAMP production and inhibition of TNF-alpha production by beta2-adrenergic stimulation, suggesting that reduced GRK activity is associated with increased sensitivity to beta2-adrenergic activation.

Cloning of GPR37, a gene located on chromosome 7 encoding a putative G-protein-coupled peptide receptor, from a human frontal brain EST library.

A cDNA sequence encoding a putative peptide-specific G-protein-coupled receptor (GPR37) was isolated from a set of human brain frontal lobe expressed sequence tags. The GPR37 cDNA predicts a single open reading frame coding for a 613-amino-acid protein with seven hydrophobic transmembrane domains. The GPR37 genomic sequence was mapped to chromosome 7q31, and it was isolated upon screening of a chromosome 7-specific genomic library. The GPR37 gene spans more than 25 kb and contains two exons and a single intron which interrupts the GPR37 cDNA within the sequence encoding the presumed third transmembrane domain. Northern blot analysis with GPR37 probes revealed a main 3.8-kb mRNA and a less abundant 8-kb mRNA, both expressed in human brain tissues, particularly in corpus callosum, medulla, putamen, and caudate nucleus. The lowest level of expression was detected in cerebellum. The 3.8-kb mRNA is also less abundantly expressed in liver and placenta. Although the ligand for the putative GPR37 receptor has not been identified, its deduced amino acid sequence shows a high degree of homology (approximately 40% in the transmembrane regions) with most mammalian peptide-specific G-protein-coupled receptors and particularly with the human endothelin-B, bombesin-BB1, and bombesin-BB2 receptors.

Molecular analysis of the functional role of beta-adrenergic receptor kinase 1 amino-terminal.

Receptor phosphorylation is a key step in the process of rapid desensitization. beta-adrenergic receptor kinase (betaARK) is a specific receptor kinase that is known to phosphorylate and induce desensitization of several G-coupled receptors only when they are occupied by their agonists. In the present study we have done several modifications to the amino-terminal of betaARK1, in order to clarify its functional role. The recombinant mutants were tested for their ability to phosphorylate rhodopsin present in purified bovine ROS membranes which serves as a substrate for betaARK1. Their expression levels were detected by Western blot analysis. We found that when the amino-terminal of betaARK1 is modified its expression level is very low, hence it is not able to phosphorylate over the basal. These findings suggest that this region is crucial for the normal processing of the protein.

Two isoforms of G protein-coupled receptor kinase 4 identified by molecular cloning.

Using PCR we found that G protein-coupled receptor kinase4 (GRK4) mRNA is expressed only in brain out of several tissues tested. In the brain two amplification products were generated. Sequence analysis revealed that the two fragments differed only by the presence or absence of an in-frame-sequence of 96 bp/32 amino acids, located near the N-terminal of the kinase. This demonstrates the existence of two isoforms of GRK4 which were named GRK4A and GRK4B in the presence or absence of the insert, respectively. This is the first evidence that, within the GRKs gene family, different isoforms do exist.

Rhodopsin phosphorylation by transiently expressed human beta ARK1: a new method for drug development.

Receptor phosphorylation is a key step in the process of rapid desensitization of the beta-adrenergic and other related G-coupled receptors. A specific kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified, which phosphorylates the agonist-occupied form of these receptors. We have cloned the cDNA for human beta ARK1. The full-length cDNA was inserted in an expression vector (pBJI neo) and used for the transfection of eukaryotic cells (COS7). The kinase activity of the cytosolic fraction of COS7 cells was assayed 72 hours after beta ARK1 transfection. A 40-70 fold increase in cytosolic beta ARK1 activity was observed. To validate this approach we demonstrated a different degree of kinase inhibition by various types of heparin. Our system, based on transient gene expression and in vitro phosphorylation of rhodopsin, represents a new method to screen for pharmacological agents acting on this kinase.