Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay.

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

Allopregnanolone activates GABA(A) receptor/Cl(-) channels in a multiphasic manner in embryonic rat hippocampal neurons.

Although 3alpha-substituted metabolites of progesterone are well established to interact with GABA(A) receptor/Cl(-) channels, the nature of the interaction(s) remains uncertain. We used patch-clamp recording to study the interaction with GABA(A) receptor/Cl(-) channels expressed by embryonic hippocampal neurons differentiating in culture and nonneuronal cells transfected with GABA(A) receptor subunits. Allopregnanolone primarily induced multiphasic current responses in neurons, which were eliminated by bicuculline, an antagonist of GABA at GABA(A) receptor/Cl(-) channels. Similar multiphasic responses blocked by bicuculline were induced by allopregnanollone in nonneuronal cells transfected with alpha(1) and gamma(2) subunits, indicating that the steroid activation of GABA(A) receptor/Cl(-) channels occurred independently of GABA. Fluctuation analyses of current responses to allopregnanolone and GABA revealed underlying channel activities with similar estimated unitary properties. However, although both agonists activated Cl(-) channels with similar estimated short and long burst-length durations, most of those stimulated by the steroid were short, while most of those opened by GABA were long. Allopregnanolone potentiated GABA-evoked Cl(-) currents in nonneuronal cells transfected with alpha(1) and beta(2) or beta(3) subunits, which did not exhibit multiphasic responses to the steroid, indicating another, independent action of the steroid at activated receptors. Pertussis toxin treatment eliminated the low-amplitude current and attenuated the high-amplitude current induced by allopregnanolone in a reversible manner. Mastoparan, which activates G proteins directly, triggered a high-amplitude current after a delay, which was blocked by bicuculline. The results indicate that allopregnanolone interacts with GABA(A) receptor/Cl(-) channels expressed by embryonic hippocampal neurons in multiple ways, some of which are mediated by G proteins.