MULTIPLE CYCLIC-AMP RECEPTORS ARE LINKED TO ADENYLYL CYCLASE IN DICTYOSTELIUM

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and galpha2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in galpha2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or galpha2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTPgammaS) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and galpha2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require Galpha2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via Galpha2. 3) Neither cAR1 nor Galpha2 is required for GTPgammaS-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.