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Cloning and expression of the A2a adenosine receptor from guinea pig brain

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Abstract

A full-length complementary DNA (cDNA) clone encoding the guinea pig brain A2 adenosine receptor has been isolated by polymerase chain reaction (PCR) and low-stringency-hybridization screening of a guinea pig brain cDNA library. This cDNA contains a long open reading frame encoding a 409-amino acid-residue protein which is highly homologous to the A2 adenosine receptors previously cloned from other species. Hydrophobicity analysis of the deduced protein sequence reveals seven hydrophobic regions, characteristic of a member of the G-protein-coupled receptor superfamily. Radioligand binding assay and functional (GTPase and cAMP) assays of the receptor, transiently expressed in mammalian cells, demonstrate typical characteristics of the A2 type adenosine receptor. The messenger RNA (mRNA) of this A2 receptor is found in the brain, heart, kidney and spleen. Receptor autoradiography with [3H]CGS21680, a specific A2 agonist, and in situ hybridization with A2 cRNA probe in guinea pig brain indicate that the receptor is expressed exclusively in the caudate nucleus. The pharmacological profile and anatomical distribution of this receptor indicate that it is of the A2a subtype. This work represents the first cloning of an A2a receptor in a rodent species, offers a complete pharmacological characterization of the receptor and provides an anatomical comparison between binding profile and gene expression of the receptor.

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Abbreviations

ADAC:

adenosine amine congener

BA:

N6-benzyladenosine

bp:

nucleotide base pair

cAMP:

cyclic adenosine 3′,5′-monophosphate

CCPA:

2-chloro-N6-cyclopentyladenosine

CGS 21680:

2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido adenosine hydrochloride

CHA:

N6-cyclohexyladenosine

CNS:

central nervous system

CPA:

N6-cyclohexyladenosine

CNS:

central nervous system

CPA:

N6-cyclopentyladenosine

CPX:

8-cyclopentyl-1,3-dipropylxanthine

DME:

Dulbecco's modified Eagle's medium

DMPX:

3,7-dimethyl-1-propargylxanthine

DPMX:

1,3-dipropyl-7-methylxanthine

DPX:

1,3-dipropyl-8-(2-amono-4-chlorophenyl)xanthine

FCS:

fetal calf serum

IBMX:

3-isobutyl-1-methylxanthine

KHB:

Kreb-HEPES buffer

MECA:

5′-N-methylcarboxamidoadenosine

NECA:

5′-N-ethylcarboxamidoadenosine

D-PBS:

Dulbecco's phosphate buffered saline

PCR:

polymerase chain reaction

R-PIA:

R(−)-N6-(2-phenylisopropyl)adenosine

SSPE:

sodium chloride-sodium phosphate-EDTA buffer

TM:

transmembrane domain

XAC:

xanthine amine congener

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Authors and Affiliations

  1. Mental Health Research Institute, The University of Michigan, 48109, Ann Arbor, Michigan

    Fan Meng, Guo-xi Xie, Derek Chalmers, Caurnel Morgan, Stanley J. Watson Jr. & Huda Akil

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  1. Fan Meng
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  2. Guo-xi Xie
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  3. Derek Chalmers
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  4. Caurnel Morgan
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Special issue dedicated to Dr. Bernard W. Agranoff

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Meng, F., Xie, Gx., Chalmers, D. et al. Cloning and expression of the A2a adenosine receptor from guinea pig brain. Neurochem Res 19, 613–621 (1994). https://doi.org/10.1007/BF00971338

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