The sigma-2 (σ2) receptor has been suggested to be a promising target for pharmacological interventions to curb tumor progression. Development of σ2-specific ligands, however, has been hindered by lack of understanding of molecular determinants that underlie selective ligand-σ2 interactions. Here we have explored effects of electron donating and withdrawing groups on ligand selectivity for the σ2 versus σ1 receptor using new benzamide-isoquinoline derivatives. The electron-donating methoxy group increased but the electron-withdrawing nitro group decreased σ2 affinity. In particular, an extra methoxy added to the para-position (5e) of the benzamide phenyl ring of 5f dramatically improved (631 fold) the σ2 selectivity relative to the σ1 receptor. This para-position provided a sensitive site for effective manipulation of the sigma receptor subtype selectivity using either the methoxy or nitro substituent. Our study provides a useful guide for further improving the σ2-over-σ1 selectivity of new ligands.

Interactions between the transducin α-subunit (Gαt) and the cGMP phosphodiesterase γ-subunit (PDEγ) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Gαt/PDEγ complexes, in particular, the signaling-state complex represented by Gαt·GTPγS/PDEγ. As a preliminary step in our effort for NMR structural analysis of Gαt/PDEγ interactions, we have developed efficient protocols for the large-scale production of recombinant Gαt (rGαt) and homogeneous and functional isotopically labeled PDEγ from Escherichia coli cells. One-step purification of rGαt was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGαt. The purified rGαt was found to be functional in GTPγS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEγ, rGαt·GTPγS/[U-13C,15N]PDEγ. The labeled PDEγ sample yielded a well-resolved 1H–15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGαt and isotope-labeled PDEγ should support further NMR structural analysis of the rGαt/PDEγ complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.

The α and β tubulins compose the microtubule cytoskeleton which is involved in many cellular processes such as vesicular transport. The photoreceptor cells in the retina are neurons specialized for phototransduction. Here we report a novel interaction between tubulin and the photoreceptor cGMP phosphodiesterase (PDE6) gamma subunit (PDEγ). The specificity and molecular details of the PDEγ:tubulin interaction were analyzed through the experiments of pull down, microtubule co-sedimentation, and NMR spectroscopy. The tubulin-interacting site was identified to be in the PDEγ C-terminal I67-G85 region, and the interaction interface appeared to be distinct from those with the other PDEγ targets in phototransduction. We also observed that PDEγ interacted with tubulin in a GTP-dependent manner. Our findings offer implications for non-phototransduction role(s) of PDEγ in the photoreceptor neurons.