Dataset Information

Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation.

ABSTRACT: In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotide-binding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the previously cloned catecholamine receptors. Northern blot analysis of various neural tissues revealed a transcript of approximately 4 kb that was predominantly located in the striatum with lesser amounts in the cortex and retina. In contrast, no mRNA was detected in the cerebellum, hippocampus, olfactory bulb, mesencephalon, or pituitary. In situ hybridization analysis also revealed a high abundance of mRNA in the striatum as well as in the olfactory tubercle. To establish the identity of this cDNA, we performed transient expression experiments in COS-7 cells. [3H]SCH-23390, a D1-selective radioligand, exhibited specific, saturable binding only in cells that were transfected with this cDNA. Competition binding analysis with a variety of dopaminergic ligands demonstrated a D1 dopaminergic pharmacology. In addition, dopamine as well as other D1-selective agonists stimulated cAMP accumulation in transfected COS-7 cells. We conclude that we have cloned a cDNA encoding the D1 dopamine receptor linked to the activation of adenylyl cyclase activity.

SUBMITTER: Monsma FJ

PROVIDER: S-EPMC54609 | biostudies-other | 1990 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation.

Monsma F J FJ Mahan L C LC McVittie L D LD Gerfen C R CR Sibley D R DR

Proceedings of the National Academy of Sciences of the United States of America 19900901 17

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotide-binding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal libra ...[more]

PMID: 2168556

Similar Datasets

Project description:Background and purposeDespite the view that only ?2- as opposed to ?1-adrenoceptors (?ARs) couple to G(i), some data indicate that the ?1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate ?2ARs (?1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

Project description:Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.

Dataset Information

Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation.

Publications

Molecular cloning and expression of a D1 dopamine receptor linked to adenylyl cyclase activation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets