The C-terminal cytoplasmic tail of polycystin-2 (PC2/TRPP2), a Ca 2+ -permeable channel, is frequently mutated or truncated in autosomal dominant polycystic kidney disease. We have previously shown that this tail consists of three functional regions: an EF-hand domain (PC2-EF, 720–797), a flexible linker (798–827), and an oligomeric coiled coil domain (828–895). We found that PC2-EF binds Ca 2+ at a single site and undergoes Ca 2+ -dependent conformational changes, suggesting it is an essential element of Ca 2+ -sensitive regulation of PC2 activity. Here we describe the NMR structure and dynamics of Ca 2+ -bound PC2-EF. Human PC2-EF contains a divergent non-Ca 2+ -binding helix-loop-helix (HLH) motif packed against a canonical Ca 2+ -binding EF-hand motif. This HLH motif may have evolved from a canonical EF-hand found in invertebrate PC2 homologs. Temperature-dependent steady-state NOE experiments and NMR R 1 and R 2 relaxation rates correlate with increased molecular motion in the EF-hand, possibly due to exchange between apo and Ca 2+ -bound states, consistent with a role for PC2-EF as a Ca 2+ -sensitive regulator. Structure-based sequence conservation analysis reveals a conserved hydrophobic surface in the same region, which may mediate Ca 2+ -dependent protein interactions. We propose that Ca 2+ -sensing by PC2-EF is responsible for the cooperative nature of PC2 channel activation and inhibition. Based on our results, we present a mechanism of regulation of the Ca 2+ dependence of PC2 channel activity by PC2-EF.
The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.
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