Fig. 3.

Rab4a- and Rab11a-mediated recycling and resensitization of CRF₁. HEK-CRF₁ cells coexpressing the wild types (wt) Rab4-GFP or Rab11-GFP, or DN mutants Rab4aN121I-GFP or Rab11aS25N-GFP, were challenged with Ucn1 or CRF (100 nm) for 30 min, washed, and placed in agonist-free medium for 4 h for recovery. A, In cells coexpressing Rab4-wt or Rab11-wt, CRF₁ recycled to the cell surface (arrowheads). B, In cells coexpressing Rab4-DN, CRF₁ also recycled to the cell surface (arrowheads). In cells coexpressing Rab11-DN, the majority of Ucn1-stimulated CRF₁ was retained in intracellular vesicles (arrows), whereas CRF-stimulated CRF₁ still recycled to the cell surface (arrowheads). Scale, 10 μm. C, Quantification of Ucn1- (red) and CRF-stimulated (blue) Ca²⁺ signals after 4 h of recovery and Rab4 dependence. CRF- but not Ucn1-stimulated Ca²⁺ signals fully resensitized in HEK-CRF₁ cells coexpressing Rab4-DN. Knockdown of Rab4 by coexpression of double-stranded RNA against Rab4 had no effects on resensitization of Ucn1- or CRF-stimulated Ca²⁺ signals. D, Rab11-dependent resensitization. CRF-stimulated, but not Ucn1-stimulated, Ca²⁺ signals fully resensitized in HEK-CRF₁ cells coexpressing Rab11-DN. Knockdown of Rab11 inhibited resensitization of Ucn1- or CRF-stimulated Ca²⁺ signals. (n ≥ 3 experiments; *, P < 0.05 compared with vehicle). ds, Double stranded.