CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending
Figure 2
CRIS is a novel target for cAMP.
(A) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). (B) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. (C) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. (D–G) Analysis of cAMP binding using FRET. (D) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. (E) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. (F) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. (G) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.