Use of nucleotide photoaffinity probes to study hormone action

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.