JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
M3 muscarinic receptor interaction with phospholipase C ?3 determines its signaling efficiency.
J. Biol. Chem.
PUBLISHED: 03-04-2014
Show Abstract
Hide Abstract
Phospholipase C? (PLC?) enzymes are activated by G protein-coupled receptors through receptor-catalyzed guanine nucleotide exchange on G??? heterotrimers containing Gq family G proteins. Here we report evidence for a direct interaction between M3 muscarinic receptor (M3R) and PLC?3. Both expressed and endogenous M3R interacted with PLC? in coimmunoprecipitation experiments. Stimulation of M3R with carbachol significantly increased this association. Expression of M3R in CHO cells promoted plasma membrane localization of YFP-PLC?3. Deletion of the PLC?3 C terminus or deletion of the PLC?3 PDZ ligand inhibited coimmunoprecipitation with M3R and M3R-dependent PLC?3 plasma membrane localization. Purified PLC?3 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). PLC?3 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 G?q-dependent but not G??-dependent PLC?3 activation. Disruption of key residues in M3Ri3N and of the PDZ ligand in PLC?3 inhibited M3Ri3-mediated potentiation. We propose that the M3 muscarinic receptor maximizes the efficiency of PLC?3 signaling beyond its canonical role as a guanine nucleotide exchange factor for G?.
Related JoVE Video
Direct-reversible binding of small molecules to G protein ?? subunits.
Biochim. Biophys. Acta
PUBLISHED: 03-25-2011
Show Abstract
Hide Abstract
Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits ?, ?, ? mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to G?? and interfere with or enhance binding of select effectors with G??. To understand the molecular mechanisms of selectivity and assess binding of compounds to G??, we used biophysical and biochemical approaches to directly monitor small molecule binding to G??. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to G?? with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the k(off) was slow for most of the compounds in physiological buffers, they could be removed from G?? with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-G?? interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of G??-effector interactions is by specific direct non-covalent and reversible binding of small molecules to G??. This is highly relevant to development of G?? targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.
Related JoVE Video
NMR analysis of G-protein betagamma subunit complexes reveals a dynamic G(alpha)-Gbetagamma subunit interface and multiple protein recognition modes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-16-2009
Show Abstract
Hide Abstract
G-protein betagamma (Gbetagamma) subunits interact with a wide range of molecular partners including: G(alpha) subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gbetagamma, a method for site-specific (15)N-labeling of Gbeta-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of (15)N-Trp Gbetagamma identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GbetaW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GbetaW99 is highly mobile on the nanosecond timescale in comparison with other Gbeta tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GbetaW99 and GbetaW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of G(alpha)(i1)-GDP to Gbetagamma had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gbetagamma subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.