Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improved plasma exposure.

Transient transfection of mammalian cells in suspension culture has recently emerged as a very useful method for production of research-scale quantities of recombinant proteins. The most commonly used cell lines for this purpose are suspension-adapted HEK and CHO cells. We report here that the plasma exposure in mice of an IL-23R extracellular domain Fc fusion protein (IL23R-Fc) differed dramatically depending on whether the protein was prepared by transient transfection of HEK or CHO cells. Specifically, IL23R-Fc expressed using CHO cells had about 30-fold higher in vivo plasma exposure compared to the HEK-expressed protein. In contrast to their differing plasma exposures, the HEK- and CHO-expressed proteins had equivalent in vitro biological activity. Characterization of the CHO- and HEK-expressed IL23R-Fc proteins indicated that the differences in in vivo plasma exposure between them are due to differential glycosylation.

Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity.

PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

Evidence for association of GABA(B) receptors with Kir3 channels and regulators of G protein signalling (RGS4) proteins.

Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein-protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 A of each other in the plasma membrane of living cells. GABA(B) R1 and R2 receptors, Kir3 channels, Galphao subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABA(B) R1 and R2 subunits ( approximately 12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABA(B) R1 or R2 subunits ( approximately 13% FRET), and between Galphao and GABA(B) R1 or R2 subunits ( approximately 10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABA(B) R1 or R2 subunits ( approximately 10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Galphao. These data are discussed in terms of a model in which GABA(B) receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

Endogenous RGS proteins enhance acute desensitization of GABA(B) receptor-activated GIRK currents in HEK-293T cells.

The coupling of GABA(B) receptors to G-protein-gated inwardly rectifying potassium (GIRK) channels constitutes an important inhibitory pathway in the brain. Here, we examined the mechanism underlying desensitization of agonist-evoked currents carried by homomeric GIRK2 channels expressed in HEK-293T cells. The canonical GABA(B) receptor agonist baclofen produced GIRK2 currents that decayed by 57.3+/-1.4% after 60 s of stimulation, and then deactivated rapidly (time constant of 3.90+/-0.21 s) upon removal of agonist. Surface labeling studies revealed that GABA(B) receptors, in contrast to micro opioid receptors (MOR), did not internalize with a sustained stimulation for 10 min, excluding receptor redistribution as the primary mechanism for desensitization. Furthermore, heterologous desensitization was observed between GABA(B) receptors and MOR, implicating downstream proteins, such G-proteins or the GIRK channel. To investigate the G-protein turnover cycle, the non-hydrolyzable GTP analogue (GTPgammaS) was included in the intracellular solution and found to attenuate desensitization to 38.3+/-2.0%. The extent of desensitization was also reduced (45.3+/-1.3%) by coexpressing a mutant form of the Galphaq G-protein subunit that has been designed to sequester endogenous RGS proteins. Finally, reconstitution of GABA(B) receptors with Galphao G-proteins rendered insensitive to RGS resulted in significantly less desensitization (28.5+/-3.2%). Taken together, our results demonstrate that endogenous levels of RGS proteins effectively enhance GABA(B) receptor-dependent desensitization of GIRK currents.

Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex.

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.

Minimal structural rearrangement of the cytoplasmic pore during activation of the 5-HT3A receptor.

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.

betaL-betaM loop in the C-terminal domain of G protein-activated inwardly rectifying K(+) channels is important for G(betagamma) subunit activation.

The activity of G protein-activated inwardly rectifying K(+) channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G(betagamma) subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying G(betagamma) activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G(betagamma) binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2(L344E) and GIRK2(G347H), that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of G(betagamma) subunits. Combining the two mutations (GIRK2(EH)) led to a more severe reduction in carbachol-activated and G(betagamma)-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2(L344E) and GIRK2(EH) also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G(betagamma)-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G(betagamma) activation. In vitro G(betagamma) binding assays revealed an approximately 60% decrease in G(betagamma) binding to the C-terminal domain of GIRK2(L344E) but no statistical change with GIRK2(EH) or GIRK2(G347H), though both mutants exhibited G(betagamma)-impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G(betagamma) activation of GIRK2 channels. Based on the 1.8 A structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the betaL-betaM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G(betagamma) alters the interaction between the betaL-betaM loop and the N-terminal domain.