Abstract

The changing relationship between stimuli and responses after prolonged receptor stimulation is a general feature of hormonal signaling systems, termed desensitization. This phenomenon has been best exemplified in the covalent modification of the G protein-linked catecholamine receptors. However, other components within this signaling pathway can be involved in desensitization. Here we present evidence that desensitization occurs at the level of the effector enzyme itself through phosphorylation. Type V adenylyl cyclase (AC) is the major isoform expressed in the heart. Using purified enzymes, we demonstrate that protein kinase A (PKA) directly phosphorylates and thereby inhibits type V AC catalytic activity. This inhibition was negated in the presence of PKA inhibitor. Analysis of enzyme kinetics revealed that this inhibition was due to a decrease in the catalytic rate, not to a decrease in the affinity for the substrate ATP. Our results indicate that AC catalytic activity can be regulated through PKA-mediated phosphorylation, suggesting another mechanism of desensitization for receptor pathways which signal via increases in intracellular cAMP.

Highlights

  • HL-21006, HL-38070 and a grant from Lederle Laboratories

  • We have demonstrated that AC itself can be regulated by phosphorylation [2]. This occurs through protein kinase C (PKC) in an isoenzyme-specific manner, and leads to an enhancement of catalytic activity

  • PKA Inactivates Type V AC Catalytic Activity-As shown in Fig. 1, incubation of purified type V AC with PKA resulted in a decrease in both basal

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Summary

Introduction

HL-21006, HL-38070 and a grant from Lederle Laboratories. The costs of publication of this article were defrayed in part by the payment of page charges. BARK presumably recognizes changes in the tertiary structure of the receptor caused by ligand binding, phosphorylating only the activated form of the receptor. In both cases receptor phosphorylation occurs only when the signaling pathway is activated; it is triggered either by agonist occupation of the receptor or by cyclic AMP formation as a result of receptor activation, forming a closed loop of negative feedback. PKA-mediated phosphorylation of the AC molecule itself and consequent changes in catalytic activity have not been clearly demonstrated; these were the goals of the present study

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