Determination of Amino Acid Residues that are Accessible from the Ligand Binding Crevice in the Seventh Transmembrane-Spanning Region of the Human A1 Adenosine Receptor
Abstract
The substituted-cysteine accessibility method (SCAM) was applied to transmembrane span seven of the human A1 adenosine receptor (hA1AR) to reveal a subset of amino acids that are exposed to the ligand-binding crevice. The SCAM approach involved a systematic probe of receptor structure by individual substitutions of residues K265 (7.30) to R296 (7.61) with cysteine. In most cases, hA1AR substituted-cysteine mutant membranes displayed antagonist dissociation binding constants that did not differ significantly from wild-type (WT). Radioligand binding assays were used to compare cell membranes that were treated with hydrophilic, sulfhydryl-specific methanethiosulfonate derivatives with control cell membranes. Position H278 was previously reported to be required for A1AR ligand binding; however, that report did not establish that H278 represents a contact point for ligands. Cysteine-substitution at H278 yields membrane preparations with greatly decreased receptor density compared with WT membranes from cells in the same transfection experiment. However, H278C membranes retain a measurable fraction of antagonist binding. This observation allows for the investigation of binding-crevice accessibility at position 278 and suggests that H278 may not be required for binding of antagonist ligands. Our data reveal the binding-crevice accessibility of residues T270 (7.35), A273 (7.38), I274 (7.39), T277 (7.42), H278 (7.43), N284 (7.49), and Y288 (7.53) in the hA1AR. These data are consistent with the high-resolution structure of bovine rhodopsin that features three α-helical turns in this region that are interrupted by an elongated, nonhelical structure from positions 7.43 to 7.48 in the primary amino acid sequence.
Footnotes
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Send reprint requests to: Dr. Jack N. Wells, Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600. Email:jack.wells{at}mcmail.vanderbilt.edu
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This work was funded through the American Heart Association Southeast Affiliate Grant-in-Aid and National Institutes of Health Grants R01-GM55813 and GM07628.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- SCAM
- substituted-cysteine accessibility method
- NECA
- 5′-N-ethylcarboxamidoadenosine
- GppNHp
- 5′-guanylylimidodiphosphate
- [3H]DPCPX
- 8-cyclopentyl-1,3-di(2,3-3H-propyl)xanthine
- MTSEA
- 2-aminoethyl methanethiosulfonate hydrobromide
- MTSET
- [2-(trimethylammonium)ethyl]methanethiosulfonate bromide
- MTSES
- sodium (2-sulfonatoethyl)methanethiosulfonate
- MTS-TEAH
- [6-(triethylammonium)hexyl] methanethiosulfonate bromide
- MTS-EDANS
- sodiumN-(methanethiosulfonylethylcarboxamidoethyl)-5-naphthylamine-1-sulfonic acid
- WT
- wild-type
- HF
- hexahistidine-FLAG-epitope-tagged
- hA1AR
- human A1 adenosine receptor
- TM
- transmembrane span
- HE buffer
- Na HEPES/Na4EDTA buffer
- KD
- dissociation binding constant
- ANOVA
- analysis of variance
- MTS-X
- methanethiosulfonate derivatives
- KI
- inhibition binding constant
- Bmax
- maximum receptor density
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- Received November 6, 2000.
- Accepted February 6, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
