BBSome tip accumulation is required for the retrieval of SSTR3 and GPR161. (A and B) PTX slows down the exit of SSTR3 (A) and GPR161 (B). IMCD3-[pEF1αΔ-^APSSTR3^NG] or IMCD3-[pCrys-^APGPR161^NG3] were pretreated with PTX for 16 h to fully inactivate Gαi. After agonist treatment, ciliary GPCR levels were measured in live cells as described in Fig. 1 (F and G). Error bars represent 95% CI. n = 10–28 cilia. (C and D) PKA inhibition accelerates the exit of SSTR3 (C) and GPR161 (D). IMCD3-[pEF1αΔ-^APSSTR3^NG] or IMCD3-[pCrys-^APGPR161^NG3] cells were treated for 3 h with the indicated concentrations of agonist and/or PKI. Ciliary level of GPCRs were measured by NG fluorescence before and after treatment to estimate the rate of exit. Addition of PKI together with subsaturating concentrations of agonist significantly accelerated the GPCR exit rates to the near-maximal values observed with saturating concentrations of agonist. Error bars represent error of the fit. n = 67–112 cilia from three independent experiments. Asterisks indicate multiple regression significance values. *, P < 0.05. (E) GPR161 retrieval can be triggered by sst treatment. IMCD3-[pCrys-^APGPR161^NG3, pEF1αΔ-^APSSTR3] cells were treated with SAG or sst for 2 h. NG fluorescence was tracked in individual cilia. Data were fitted to a single exponential. n = 10–21 cilia. (F) SAG treatment is not sufficient to trigger the retrieval of SSTR3. IMCD3-[pEF1αΔ-^APSSTR3^NG] cells were treated with SAG for 2 h. ^APSSTR3 was pulse-labeled with mSA647 and individual cilia were tracked. Data were fitted to a single exponential. Error bars represent 95% CI. n = 12 cilia.