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. 2018 Dec 6;160(2):276–291. doi: 10.1210/en.2018-00920

Figure 5.

Figure 5.

Effects of histidine residue targeted mutations in CRHR1 on the AtT-20 acid response. (A) Homology of CRHR1 in human, mouse, rat, and sheep. Boxes indicate conserved histidine residues. (B) Schematic of histidine residues in mouse Crhr1 with histidine residues highlighted. Membrane topology model of Crhr1 was generated by Protter version 1.0. (C) Immunocytochemistry of Crhr1-KO cells transfected with Crhr1-pMF, histidine residue mutated Crhr1-pMF (H115F, H117F, H127F, H155F, H181F, H199F, and H214F), or pMF (empty vector) with the antibody for CRHR1 (green, Crhr1; blue, 4′,6-diamidino-2-phenylindole). (D) Western blots for Crhr1 in Crhr1-KO cells transfected with Crhr1-pMF, histidine residue mutated Crhr1-pMF (H115F, H117F, H127F, H155F, H181F, H199F, and H214F), or pMF (empty vector). (E) cAMP levels with (+) or without (−) 10−7 M CRH in Crhr1-KO AtT-20 cells transfected with Crhr1-pMF, histidine residue mutated Crhr1-pMF, or pMF (empty vector). Results are mean ± SD. **P < 0.01 vs no CRH. (F) Quantitative RT-PCR analysis of Pomc and Gh gene expression in Crhr1-KO AtT-20 cells transfected with Crhr1-pMF (WT), histidine residue mutated Crhr1-pMF, or pMF (KO) after 24-h treatment in normal pH 7.4 (N) and acidic pH 6.8 (A) medium. (G) Western blots for Pomc, CamkII, p-CamkII, and β-actin (upper panels) and quantification of protein levels (lower panels) in Crhr1-KO AtT-20 cells transfected with Crhr1-pMF, histidine residue mutated Crhr1-pMF, or pMF after 24-h treatment in normal pH 7.4 (N) and acidic pH 6.8 (A) medium. Results are mean ± SD. *P < 0.05 vs pH 7.4; **P < 0.01 vs pH 7.4. n.s., not significant.