Alan Kosaka

Cloning and expression of a 5-hydroxytryptamine7 receptor positively coupled to adenylyl cyclase.

Abstract: A cDNA clone (designated as GP2‐7) encoding a novel 5‐hydroxytryptamine (5‐HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5‐HT receptor subtypes (34–48%). The sequence of GP2‐7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5‐HT7. mRNA for GP2‐7 was detected in cortical and limbic brain regions. Transiently expressed GP2‐7 showed high‐affinity binding to [3H]5‐HT (pKi = 9.0) with the following rank order of affinities: 5‐carboxyamidotryptamine (5‐CT) > 5‐HT = 5‐methoxytryptamine (5‐MeOT) > methiothepin > 8‐hydroxy‐2‐(dipropylamino)tetralin (8‐OH‐DPAT) > spiperone ≫ sumatriptan. Adenylyl cyclase activity in CHO‐K1 cells transiently transfected with GP2‐7 was stimulated by several analogues of 5‐HT with the following order of potency: 5‐CT > 5‐HT = 5‐MeOT > dipropyl‐5‐CT > 8‐OH‐DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2‐7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5‐CT.

Cloning, expression and pharmacology of a truncated splice variant of the human 5‐HT7 receptor (h5‐HT7(b))

1 The rat 5‐hydroxytryptamine (5‐HT)7 receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy‐terminus. The human 5‐HT7 receptor gene contains at least two introns and encodes a 445 amino acid 5‐HT receptor. 2 A truncated splice variation in the human 5‐HT7 receptor was isolated from a human placental cDNA library. In accordance with current NC‐IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5‐HT7(b) receptor and the long form of the receptor as h5‐HT7(a). 3 The h5‐HT7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [3H]‐5‐carboxyamidotryptamine (5‐CT; Kd=0.28±0.06 nM, Bmax=7.3±1.7 pmol mg−1 protein). The rank order of affinities (pKi) for a series of ligands was: 5‐carboxamidotryptamine (5‐CT, 9.65)>5‐hydroxytryptamine (5‐HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8‐hydroxy‐2(di‐n‐propylamino)tetralin (8‐OH‐DPAT, 6.85)>ketanserin (6.44). 4 The h5‐HT7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC50): 5‐CT (8.7±0.11)>5‐MeOT (5‐methoxytryptamine; 8.1±0.20)>5‐HT (7.5±0.13)>tryptamine (5.6±0.36)>8‐OH‐DPAT (5.3±0.28)>5‐methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies. 5 In a similar fashion to that described for the 5‐HT7(a) receptor, PCR studies suggested that the 5‐HT7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta. 6 It is concluded that the h5‐HT7 receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5‐HT7 receptor gene.

Molecular cloning, genomic characterization and expression of novel human α1A-adrenoceptor isoforms

We have isolated and characterized from human prostate novel splice variants of the human α1A‐adrenoceptor, several of which generate truncated products and one isoform, α1A‐4, which has the identical splice site as the three previously described isoforms. Long‐PCR on human genomic DNA showed that the α1A‐4 exon is located between those encoding the α1A‐1 and α1A‐3 variants. CHO‐K1 cells stably expressing α1A‐4 showed ligand binding properties similar to those of the other functional isoforms as well as agonist‐stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that α1A‐4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

RG7128 Alone or in Combination with Pegylated Interferon-α2a and Ribavirin Prevents Hepatitis C Virus (HCV) Replication and Selection of Resistant Variants in HCV-Infected Patients

INTRODUCTION RG7128 (prodrug of PSI-6130) shows potent antiviral efficacy in patients infected with hepatitis C virus (HCV) genotypes 1, 2, or 3, with mean viral load decreases of 2.7 and 5 log(10) IU/mL, respectively, associated with 1500-mg doses twice daily after monotherapy for 2 weeks and with 1000-mg and 1500-mg doses twice daily after treatment in combination with the standard of care (SOC) for 4 weeks. RESULTS From 32 patients treated with RG7128 monotherapy for 2 weeks, marginal viral load rebound was observed in 3 HCV genotype 1-infected patients, whereas partial response was observed in 2 genotype 1-infected patients. From 85 patients receiving RG7128 in combination with SOC, 1 HCV genotype 1-infected patient experienced a viral rebound, and 2 genotype 3-infected patients experienced a transient rebound. Five genotype 1-infected patients had an HCV load of >1000 IU/mL at the end of 4-week treatment. No viral resistance was observed, per NS5B sequencing and phenotypic studies. PSI-6130 resistance substitution S282T needs to be present at levels of ≥90% within a patients quasispecies to confer low-level resistance. No evidence of S282T was found by population or clonal sequence analyses. CONCLUSIONS The requirement for a predominant S282T mutant quasispecies, its low replication capacity, and the low-level resistance it confers probably contribute to the lack of RG7128 resistance observed in HCV-infected patients.

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway.

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.