M3 muscarinic receptor interaction with phospholipase C beta3 determines its signaling efficiency

Contains fulltext : 133821.pdf (publisher's version ) (Open Access)Phospholipase Cbeta (PLCbeta) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on Galphabetagamma heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLCbeta3. Both expressed and endogenous M3R interacted with PLCbeta in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLCbeta3. Deletion of the PLCbeta3 C terminus or deletion of the PLCbeta3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLCbeta3 plasma membrane localization. Purified PLCbeta3 bound directly to glutathione S-transferase (GST)-fused M3R intracellular loops 2 and 3 (M3Ri2 and M3Ri3) as well as M3R C terminus (M3R/H8-CT). PLCbeta3 binding to M3Ri3 was inhibited when the PDZ ligand was removed. In assays using reconstituted purified components in vitro, M3Ri2, M3Ri3, and M3R/H8-CT potentiated Galphaq-dependent but not Gbetagamma-dependent PLCbeta3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLCbeta3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLCbeta3 signaling beyond its canonical role as a guanine nucleotide exchange factor for Galpha

Structure, function, and dynamics of the G alpha binding domain of Ric-8A.

Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (G alpha). We report the 2.2-angstrom crystal structure of the Ric-8A G alpha-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440. Residues 1-429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen- deuterium exchange data indicate repeats 3 through 6 to be a putative G alpha-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1-4, 5-6, and 7-9. 2D H-1-N-1(5)-TROSY spectra of [H-2, N-15]-labeled Gail in the presence of R452 reveals chemical shift perturbations of the C terminus and Gail residues involved in nucleotide binding