Abstract
Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.
Highlights
Three main pathways havebeen implicated in desen- ogous when triggered by activation of other receptors that sitization of receptors that stimulate adenylylcyclase increase intracellular cAMP (1, 2, 7)
When cAMP was increased by type 2 vasopressin receptor can be studied under well contreatment with 10 NM PGEl and isobutylmethylxan- trolled conditions, we have investigated the types of desensithine, desensitization of the PGE, receptor was ob- tizing response it undergoes in the HTB-2cell
To characterize the desensitizing response in HTB-2 cells added and the cells incubated for 10 min at 37 "C to allow for we first determined the binding propertiesof the human V2
Summary
Ltk- cells were cotransfected (14) with plasmid pHSV-106, which is Partial Purification ofCell Membranes-After treatments in the a pBR322-based plasmid containing theherpes simplex virus thymi- absence and presence of AVP or PGE, plus IBMX confluent cells dine kinase gene and the hamster &adrenergic receptor (15), sub- from three 15-cm dishes were used per preparation. General Strategies of Experiments-Cells were treated in the ab- The pellet was resuspended with the help of a Dounce homogenizer sence and presence of test substances such as AVP, PGE,, IBMX, in 1 ml of ice-cold HE buffer containing 27% sucrose, aliquoted, washed, and tested for receptor binding, cAMP accumulation, and/ frozen, and stored at -70 "C until itwas used. Treatments of cells with PGE, plus IBMX were terminated by by the addition of 100 pl of a solution containing[40] mM ATP, 10 mM rinsing the cells twice with ice-cold D-PBS supplemented with 1mg/ CAMP, and 1%sodium dodecyl sulfate.
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