Parvathi Nuthulaganti

CLONING AND CHARACTERIZATION OF A HUMAN ANGIOTENSIN II TYPE 1 RECEPTOR

A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.

BacMam Recombinant Baculoviruses in G Protein–Coupled Receptor Drug Discovery

With completion of the sequencing of the human and mouse genomes, the primary sequences of close to 400 non-olfactory G protein-coupled receptors (GPCRs) have been determined. There are intensive efforts within the pharmaceutical industry to discover and develop new therapeutic agents acting via GPCRs. In addition, there is a concerted effort to identify potential new drug targets from the remaining 150+orphan GPCRs through the identification of their ligands. Access to functionally expressed recombinant receptors underpins both of these key drug discovery activities. Typically, GPCR drug discovery screening activities are carried out using mammalian cell lines stably expressing the target of interest. The influx of new receptor sequences originating from genomic sequencing efforts has caused a shift toward wider applications of transient rather than stable expression systems, especially in support of assays for orphan receptor ligand screening. Recombinant baculoviruses in which the polyhedrin promoter has been replaced with a mammalian promoter, termed BacMam viruses, were originally designed as potential new gene therapy delivery vehicles. This same technology offers numerous advantages as a transient expression system in the assay of membrane-expressed drug targets, including GPCRs. Data presented show that BacMam can be used rapidly to generate robust and pharmacologically authentic GPCR assays in several formats, with the potential to transform drug discovery screening processes for this gene family.

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems

1 Several peptidic urotensin‐II (UT) receptor antagonists exert ‘paradoxical’ agonist activity in recombinant cell‐ and tissue‐based bioassay systems, likely the result of differential urotensin‐II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT‐HEK (human embryonic kidney) cells. 2 BacMam‐mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U‐II>urantide ([Pen5‐DTrp7‐Orn8]hU‐II4–11)>SB‐710411 (Cpa‐c[DCys‐Pal‐DTrp‐Lys‐Val‐Cys]‐Cpa‐amide)≫GSK248451 (Cin‐c[DCys‐Pal‐DTrp‐Orn‐Val‐Cys]‐His‐amide) (the relative coupling efficiency of recombinant HEK cells was cat>human≫rat UT receptor). 3 The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low‐efficacy agonists appear to function as antagonists. 4 In contrast to the ‘low‐efficacy agonists’ urantide and SB‐710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4–5‐fold less than seen with urantide). Since GSK248451 (1 mg kg−1, i.v.) blocked the systemic pressor actions of exogenous U‐II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U‐II in the aetiology of mammalian cardiometabolic diseases.

Heterologous Expression of G Protein–Coupled Receptors in U-2 OS Osteosarcoma Cells

Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.

Cloning and Characterization of a Novel Integrin β3Subunit

We have identified a novel integrin β3 subunit, termed β3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3′-untranslated region of the β3C subunit differs from the previously reported β3A (platelet) and β3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The β3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the β3A gene. HEK 293 cells were stably co-transfected with αV and either β3C (HEKβ3C) or β3A(HEKβ3A). The viability of HEKβ3C cells was lower than that of HEKβ3A cells, and HEKβ3Ccells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both αVβ3A and αVβ3Cisoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKβ3A, HEKβ3C cells failed to adhere to osteopontin, an αVβ3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the β3 integrin in cell adhesion and suggest a potential role for the β3C integrin subunit in modulating cell-matrix interactions.

BacMam: Versatile Gene Delivery Technology for GPCR Assays

BacMam viruses are modified baculoviruses that contain mammalian expression cassettes for viral gene delivery and transient expression in mammalian cells. They are easily, inexpensively, and rapidly generated and provide a versatile solution for G protein-coupled receptor (GPCR) cell-based assay development. Using BacMam technology, target gene expression levels are easily controlled and simultaneous delivery of multiple genes is possible, for example, coexpression of a receptor and a G protein or a reporter gene. BacMam viruses are compatible with the GPCR cell-based assay formats typically used in high-throughput screening and provide an unparalleled level of experimental flexibility that is simply not possible when using stable recombinant cell lines.

Molecular and pharmacological characterization of the murine tachykinin NK3 receptor

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

M3 muscarinic acetylcholine receptor antagonists: SAR and optimization of bi-aryl amines

Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M(3) muscarinic acetylcholine receptor antagonists such as 14 (pA(2)=11.0) possessing good sub-type selectivity for M(3) over M(2). The structure-activity relationships (SAR) and optimization of the bi-aryl amine series are described.

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.

Cloning and characterization of a rabbit ortholog of human Gα16 and mouse Gα15

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G‐protein α subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Gα16 and mouse Gα15, respectively. The rabbit Gα subunit cDNA was subcloned into a mammalian expression vector and transiently co‐transfected into HEK‐293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G α subunit behaved similarly to Gα15 or Gα16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit Gα subunit appears to be the ortholog of human Gα16 and mouse Gα15.