Photoaffinity labeling of cyclic-AMP- and AMP-binding proteins differentiating Dictyostelium discoideum cells

Abstract

Cyclic-AMP-binding proteins play important roles during the differentiation of the cellular slime mold Dictyostelium discoideum. The photoaffinity reagent 8-N3-cyclic [32P]AMP has been used to label developmentally regulated cyclic-AMP-binding proteins of intact cells, membranes, and cytoplasm. 8-N3-Cyclic AMP is a chemoattractant for differentiated D. discoideum cells and is a substrate for the membrane phosphodiesterase (mPDE). When mPDE is inhibited, the only specifically labeled protein on intact cells has a molecular weight of 40,000 on sodium dodecyl sulfate gels. The developmental time course of appearance of this protein and its high specificity for cyclic AMP identify it as the cell surface chemotactic receptor for cyclic AMP. The concentration dependence of labeling of this protein is consistent with the measured chemotactic potency of 8-N3-cyclic AMP, which is about 1/100th that of cyclic AMP. Three developmentally regulated proteins (Mr 26,000, 33,000, and 36,000) of the soluble fraction (cytoplasm) are labeled by the photoaffinity reagent and are specific for cyclic AMP. By analogy with other systems, these may be regulatory subunits of protein kinases. The mPDE of ghosts or plasma membrane fractions converts the reagent to 8-N3-[32P]AMP, which specifically photoaffinity labels a protein of Mr 42,000 associated with the cytoplasmic face of the plasma membrane.