Biased Gs Versus Gq Proteins and β-Arrestin Signaling in the NK1 Receptor Determined by Interactions in the Water Hydrogen Bond Network

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and p-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved Asp11:10 (2.50). Here, we find that this Glu11:10 occupies the space of a putative allosteric modulating Na ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between (=dull: 10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) p-arrestin only; and 3) Gq and p-arrestin but not through Gs. Interestingly, increased constitutive Gs but not G signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), 111:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans- membrane receptor conformations activating different signal transduction pathways.