ABSTRACTkinases can mediate about the same maximal extent of desensitization. When the desensitization is measured as the loss of signal-transduction capacity of the receptor system (9), the action ofeach ofthe two kinases individually can result in about 50o desensitization. However, PKA-mediated desensitization occurs at about 100-fold lower agonist concentrations than p8ARK-mediated desensitization. Similar conclusions regarding the high agonist sensitivity of PKA-mediated desensitization have been drawn from studies using S49 mouse lymphoma cells defective in PKA (10).In situations where high agonist concentrations are present, such as within the synaptic cleft, the sensitivity of the desensitization response to low agonist concentrations would seem to be of little importance. Instead, the speed at which desensitization is achieved would appear to be more critical. Thus, the most rapid mechanism will be the most important in these systems. The present study describes a kinetic analysis of the three rapid mechanisms leading to ,8-receptor desensitization. Since thus far the critical amino acids of the receptor required for sequestration have not been identified and since, therefore, receptor sequestration cannot be abolished by the mutagenesis approach, we used the technique of inhibition of the three mechanisms by inhibitors (8). The inhibitors used were heparin for BARK (8, 11), the PKA inhibitor peptide (PKI) of the rabbit muscle protein inhibitor for PKA (12), and concanavalin A (Con A) pretreatment of cells for sequestration (13). Although these inhibitors are clearly not entirely specific, we have shown previously (8) that they are specific in the context of P-receptor desensitization-i.e., all of them affect only one of the three rapid desensitization mechanisms. MATERIALS AND METHODSPermeabilization of A431 Cells. Human epidermoid carcinoma A431 cells were grown to -95% confluency in Dulbecco's modified Eagle's medium supplemented with 10%Abbreviations: BARK, B-adrenergic receptor kinase; PKA, protein kinase A; PKI, PKA inhibitor peptide. 6201The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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