Density gradient profiles of A1 adenosine receptors labeled by agonist and antagonist radioligands before and after detergent solubilization.

A1 adenosine receptors in bovine cerebral cortex have been solubilized and subjected to sedimentation analysis using sucrose density gradient centrifugation. Because the receptors bound both agonists and antagonists with high affinity after solubilization, receptors labeled with an agonist or an antagonist radioligand could be studied before solubilization, after solubilization but before sucrose gradient centrifugation, or after sucrose gradient centrifugation. In each instance the agonist radioligand 125I-N6-p-aminobenzyladenosine (125I-ABA)-labeled receptor migrated as a single symmetrical peak that was located in the same area of the gradient. In contrast, the location of the receptor labeled with the antagonist [3H]xanthine amine congener [( 3H]XAC) varied in the different types of samples. When membranes were incubated with radioligands before solubilization, the peak of antagonist-labeled receptor was symmetrical and was located at a lower density than the peak of agonist-labeled receptor. In addition, receptors incubated with antagonist before solubilization migrated with an apparent lower density than receptors labeled with antagonist either after solubilization or after density gradient centrifugation. Treatments with agents that alter receptor/G protein interactions also resulted in a shift of antagonist-labeled receptors to lower density. These results suggest that the receptors that migrate to the lower density fractions of the gradients are free receptors, whereas those that migrate to the higher density fractions are coupled to a G protein. It is hypothesized that a large proportion of A1 receptors exist in the membrane coupled to a G protein and that this is the species labeled by the agonist radioligand 125I-ABA. It is, furthermore, hypothesized that the antagonist radioligand [3H]XAC preferentially binds to the free uncoupled A1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)