Factors Determining the Specificity of Signal Transduction by Guanine Nucleotide-binding Protein-coupled Receptors

To define the integration of multiple signals by different types of adenylyl cyclase (AC) within the cell, we altered the population of enzymes expressed in the cell and determined the subsequent processing of stimulatory and inhibitory input. DDT 1-MF2 cells expressed AC VI‐IX and were stably transfected with AC II, III, or IV. Enzyme expression was confirmed by RNA blot analysis and functional assays. Basal enzyme activity was only increased in AC II transfectants (6-fold). Maximum stimulation of enzyme activity was increased in each of the AC transfectants to varying extents. a2A/D-AR activation elicited enzyme type-specific responses. a2-AR activation inhibited the effect of isoproterenol in control transfectants, and this action was magnified in AC III transfectants. In AC II and AC IV transfectants, a2-AR activation initiated both positive (Gbg) and negative signals (G ia) to the Gsa-stimulated enzyme, and both types of signals were blocked by cell pretreatment with pertussis toxin. The negative input to AC II from the a2-AR was blocked by protein kinase C activation in AC II transfectants, but it was the positive input to AC IV that was compromised by protein kinase C activation. These data indicate that the integration of multiple signals by adenylyl cyclases is a dynamic process depending upon the enzyme type and phosphorylation status. Nature has evolved several clever mechanisms for cells to process external stimuli. Such systems incorporate a receptor at the cell surface that is activated by the stimulus and initiates signal propagation to the cell interior, recruiting several additional proteins in the process. Many of the groups of proteins involved in signal propagation exist as isoforms or closely related subtypes, as is the case for signals initiated by Gprotein-coupled membrane receptors. These receptors are coupled to a variety of effectors including adenylyl cyclases, phospholipases, and protein kinases. Each of these effectors exists as isoforms with different regulatory properties allowing complex signal integration, and this capability presents opportunities for the cell to engineer highly specific responses to an external stimulus. Such appears to be the case for signals involving the enzyme adenylyl cyclase. There are nine types of adenylyl cyclase (AC) 1 that differ in their regulatory mecha