Photoaffinity labeling of adenylyl cyclase

Photoaffinity is an effective tool to covalently tag amino acid residues that are in or near ligand-binding sites. The polypeptide chains that contribute to the binding site of a given receptor can be identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) if they are specifically labeled with a radioactive marker. Purification and subsequent N-terminal sequencing of these labeled peptides identify their location in the primary sequence of the receptor. Forskolin, a naturally occurring diterpene, has direct stimulatory effects on adenylyl cyclase. This chapter discusses the synthesis and use of two forskolin photoaffinity labels. N-13-(4-azido-3-[ 125 I]iodophenyl) propionyl]forskolin ( 125 I-6-AIPP-Fsk) and 125 I-7-AIPP-Fsk. Although both compounds bind to adenylyl cyclase, 125 I-6-AIPP-Fsk is a more effective photolabel for derivatization of adenylyl cyclase.