Two different photoaffinity labels, 125-I-6-AIPP-forskolin and 125-I-7- AIPP-forskolin, have been synthesized and used for studying several different membrane bound proteins: adenylyl cyclase; the glucose transporter; and the P-glycoprotein. The proteins are similar in that they have 12 transmembrane helices, bind nucleotides, and interact with forskolin, a naturally occurring drug. Binding studies with the nonradioactive derivatives of forskolin indicated that 6-AIPP-forskolin is much more specific for adenylyl cyclase than 7-AIPP-forskolin. These results concur with photoaffinity labeling experiments. 125-I-6-AIPP- forskolin photolabels adenylyl cyclase in crude bovine brain membranes and in partially purified preparations. There is no detection of photolabeling of adenylyl cyclase by 125-I-7-AIPP-forskolin. The specificity of photolabeling by 125-I-6-AIPP-forskolin was demonstrated by competition of photolabeling by forskolin, an active analog of adenylyl cyclase, but not 1,9-dideoxyforskolin, an inactive analog. 125-I-7-AIPP-forskolin photolabeled the glucose transporter. In both crude bobine brain membranes and human erythrocyte membranes, Cytocholasin B and D-glucose, inhibitors of the glucose transporter, specifically competed for the 125-I-7-AIPP- forskolin photolabeling. 125-I-6-AIPP-forskolin and 125-I-7-AIPP-forskolin photolabel the P-glycoprotein. Photolabeling is inhibited by forskolin, 1,9-dideoxyforskolin, and other calcium channel blockers specific for the P-glycoprotein. Studies are underway to localize the binding site for forskolin in the P-glycoprotein. Studies are underway to localize the binding site for forskolin in the P-glycoprotein. These photolabels are also being used to detect forskolin binding sites on other membrane transport proteins.
