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Abstract
The ability of guanine nucleotides to lower agonist binding affinity provides a convenient
indication of receptor-G protein coupling: guanine nucleotides convert muscarinic
receptors from high-affinity states for agonists to low-affinity states. We studied
the influence of assay temperature on the demonstration of this coupling in rat brainstem
and atrium. Agonist affinity of brainstem receptors increased as temperature was lowered,
reflecting a greater proportion of receptors in high-affinity conformations. The influence
of 5'-guanylylimidodiphosphate, a stable analog of GTP, on agonist binding, determined
directly (using [3H]oxotremorine-M) or indirectly (in [3H]N-methylscopolamine/carbamylcholine
competition studies), was greatest from 16 to 20 degrees. Guanine nucleotide sensitivity
was much reduced at 0-4 degrees and 37 degrees. Brainstem and atrial muscarinic receptors
were similarly affected by temperature. We suggest that high-affinity receptor-G protein
complexes are unstable at high temperatures, thereby decreasing agonist affinity and
masking the guanine nucleotide effect. At low temperatures, the receptor-G protein
complex is stabilized and fails to dissociate in the presence of guanine nucleotides.
The optimum temperature for monitoring receptor-G protein interactions in binding
assays was 16-20 degrees.