P2Y₁ receptor desensitization is mediated through a PKC-dependent but GRK2- and GRK6-independent mechanism. Cells stably transfected with P2Y₁ purinergic receptor construct were transiently transfected with pcDNA3-^K220RGRK2 (GRK2-DNM), pcDNA3-^K215RGRK6 (GRK6-DNM), vector alone (control), or scrambled GRK2- or GRK6-specific siRNA constructs. Cells were incubated in the absence or presence of the PKC inhibitors GF109203X (2 μM), Ro317549 (1 μM), or Gö6976 (1 μM) for 15 minutes before experiments. Desensitization was assessed by comparing calcium responses with those of ADP (0.1 μM) before or after a pretreatment addition of ADP (1 μM; 2 minutes), as detailed in “Materials and methods.” Values represent the mean ± SEM of 4 independent experiments. (A) Expression of GRK2-DNM (○) and GRK6-DNM (▿)in P2Y₁-expressing 1321N1 cells enhances endogenous β₂-adrenoceptor activity by decreasing the rate of desensitization of the receptor. Isoproterenol (1 μM)–induced cAMP accumulation (0-10 minutes) was assessed in 1321N1 cells. ⬡ indicates vector alone. Data represent mean ± SEM of 4 independent experiments and are expressed as pmol cAMP per well. (B) Fura-2AM–loaded cells were sequentially treated with ADP (0.1 μM) (left column of each triplet; ▪) to act as the control response, then with a desensitizing concentration of ADP (1 μM; middle column of each triplet; ▪), followed by a further test dose (0.1 μM ADP; right column in each triplet; ▪). Asterisks show responses where there is significant desensitization (right column compared with left column of each triplet; P < .05, Mann-Whitney U test). (C) Results are expressed as the response to 0.1 μM ADP after a desensitizing dose (1.0 μM ADP) as a percentage of the control response to 0.1 μM ADP. Asterisks indicate where responses are significantly desensitized relative to controls (P < .05, Mann-Whitney U test). (B-C) Data are mean ± SEM (n = 3). (D) P2Y₁ receptor desensitization is mediated in part by PKC in human platelets. Platelets were pretreated with the PKC inhibitors GF109203X (2 μM), Ro317549 (1 μM), or Gö6976 (1 μM) or with the PKC activator PMA (100 nM; 15 minutes). Pretreatment of platelets with any of these agents induced no change in cytosolic calcium (data not shown). Platelets were treated with ADP (1 μM; 30 seconds) and then with 0.2 U/mL apyrase for another 180 seconds, and they were restimulated with 10 μM ADP. Receptor desensitization was assessed by comparing a percentage of desensitization of peak calcium responses to controls that had been treated identically except for the lack of pretreatment with desensitizing ADP. Values are mean ± SEM (n = 3), and results are expressed as the ADP response after a desensitizing dose as a percentage of the control response. Asterisks indicate statistical significance at P < .05 for data compared with ADP pretreatment without PKC activator or inhibitor (control; Mann-Whitney U test).