Identification of Domains of the Human A Adenosine Receptor That Are Important for Binding Receptor Subtype-selective Ligands Using Chimeric A/A Adenosine Receptors

doi:10.1074/jbc.270.35.20485

Identification of Domains of the Human A Adenosine Receptor That Are Important for Binding Receptor Subtype-selective Ligands Using Chimeric A/A Adenosine Receptors (*)

From the Herman B Wells Center for Pediatric Research, Pediatric Endocrine Unit, James Whitcomb Riley Hospital and the Department of Biochemistry and Molecular Biology and the Program of Neurobiology, Indiana University School of Medicine, Indianapolis, Indiana 46202

^§ To whom correspondence should be addressed:
Riley Hospital, Rm. 5984, 702 Barnhill Dr., Indianapolis, IN 46202.
Fax: 317-274-5378; srivkee{at}Indyvax.IUPUI.edu

Abstract

To provide new insights into the regions of the human A¹ adenosine receptor (A¹AR) involved in ligand binding, a series of chimeric human A¹ and rat A adenosine receptors (A¹/A) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the AAR agonist [³H]CGS-21680, the A¹AR agonist [³H]2-chloro-N⁶-cyclopentyladenosine (CCPA), and the A¹AR antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the AAR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the A¹AR (A¹TM1/A), [³H]CGS-21680 and [³H]CCPA binding was detectable. When an A¹TM1-2/A construct was studied, [³H]CGS-21680 binding was lost and [³H] DPCPX binding appeared. Saturation studies using [³H]CCPA revealed that the A¹TM1/A construct had low affinity. However, with the subsequent addition of A¹AR TMs 2-4 receptor affinity improved markedly. Saturation studies using [³H]DPCPX also revealed that the TMs 1-4 of the A¹AR conferred wild-type receptor affinity. When the ligand binding properties of A¹TM1-4/A, A¹TM1-6/A, and wild type A¹AR constructs were directly compared, no differences were found using 10 different compounds. When truncated A¹ARs that extended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data suggest that the important determinants for A¹AR agonist and antagonist binding and ligand specificity are present in TMs 1-4.

Footnotes

↵* This work was supported by a Grant-in-aid from the American Heart Association, the James Whitcomb Riley Memorial Association, and National Institutes of Health Grant RO1NS326224. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank [GenBank]and X68485[GenBank].
↵¹ The abbreviations used are:

AR

adenosine receptor

TM

transmembrane

PCR

polymerase chain reaction

CCPA

2-chloro-N⁶-cyclopentyladenosine

DPCPX

8-cyclopentyl-1,3-dipropylxanthine

3′

3′-deoxyadenosine

5MT

5′-deoxy-5′-methyl-thioadenosine

CSC

8-(3-chlorostylryl)caffeine

DMPA

N⁶-[2-(3,5-dimetoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine

NECA

5′-N-ethylcarboxamidoadenosine: ANOVA, one way analysis of variance.
↵² S. A. Rivkees, unpublished results.
- Received June 2, 1995.