Fowler, Daniel J.

Thompson, Lynmarie K.

Weis, Robert M.

Elsevier - Publisher Connector 

Monday, February 22, 2010 291a(565KKKRRK570) immediately following helix 8 in the c-terminus of M3R
was found to be necessary for the decrease in Gq-V mobility. We tested the
hypothesis that an electrostatic interaction was involved in the interaction
between M3Rs and Gq by repeating our FRAP experiments in permeabilized
cells exposed to buffer solutions with high and low ionic strength. High ionic
strength solutions inhibited the decrease in Gq mobility, whereas low ionic
strength solutions enhanced this effect. These results suggest that an electro-
static interaction mediates an interaction between the c-terminus of M3Rs and
Gq heterotrimers. The functional significance of this interaction is currently
under study.
Supported by grant GM078319 from the National Institutes of Health.
1519-Pos
The Human Muscarinic Receptor Couples to GaI3 Via Catalytic Collision
Shai Berlin, Daniel Yakubovich, Tal Keren-Raifman, Nathan Dascal.
Tel Aviv University, Ramat Aviv, Tel Aviv, Israel.
Hundreds of G-protein coupled receptors (GPCR) are encoded in the human
genome. All GPCRs react to a vast variety of ligands and initiate the G-protein
activation cycle, by catalyzing the exchange of GDP by GTP on the Ga sub-
unit. Classically, this mode of activation has been proposed to be of catalytic
collision coupling nature, where a single receptor sequentially activates sev-
eral G-proteins. However, recent biophysical and imaging studies challenged
this concept and suggested that some GPCR and G-proteins form stable non-
dissociating complexes prior to and after activation. We were interested in
determining the mode of coupling between the human muscarinic 2 receptor
(m2R) and Gai3bg. We used the G-protein activated Kþ channel (GIRK)
as a reporter for receptor activation and systematically quantified receptor’s
and channel’s plasma membrane concentrations using fluorescent methods
and radioligand assays. We found a decrease in activation time at high recep-
tor density, with no change in channel concentration. However, maximal am-
plitude was attained at lower receptor density, suggesting an amplification
process. No change in Gbg concentration was observed, as judged by the un-
changed Gbg-dependent basal activity of GIRK. Additionally, increasing
amounts of m2R did not increase Gai concentration. Together, these results
suggest a catalytic collision coupling mechanism. We constructed a model de-
scribing m2R’s activation scheme and predicted that excessive Ga subunits
should slow the activation process by occupying the activated receptor in
‘‘dead-end’’ interactions, not leading to channel activation. Increasing
amounts of two fluorescent Gai3 subunits were used to test the prediction.
Indeed, both subunits slowed the evoked-current, without change in current
amplitude. These results, together with our previous observations, suggest
that the m2R activates Gai3 via a catalytic collision coupling mechanism,
where one receptor diffuses and activates several Gabg subunits, leading to
the activation of GIRK.
1520-Pos
Structural Characterization of the N-terminal Region of the Saccharomy-
ces Cerevisiae G-Protein Coupled Receptor, Ste2P
Stephanie Kendall1, Chunhua Shi2, Shelley Forgeron3, Michele C. Loewen1.
1University of Saskatchewan, Saskatoon, SK, Canada, 2Carleton University,
Ottawa, ON, Canada, 3National Research Council of Canada, Saskatoon, SK,
Canada.
Binding of a-factor pheromone to the G-protein coupled receptor, Ste2p,
initiates signal transduction events that lead to mating of the yeast Saccharo-
myces cerevisiae. Recent indirect evidence also implicates the N-terminal
region of Ste2p in modulating cell wall degradation and membrane fusion
during later steps of mating. Toward deciphering mechanisms, structural stud-
ies have been initiated on the N-terminus of Ste2p. Initially, residues 1-71 of
Ste2p were expressed as a fusion protein with HIS and KSI tags and affinity
purified from E.coli in mg quantities. Subsequent cyanogens bromide cleavage
at methionines yielded a hydrophobic peptide (Ste2p 2-54) that consistently
disappeared upon HPLC enrichment. Similarly, a chemically synthesized frag-
ment corresponding to Ste2p residues 14-43 could not be purified by HPLC.
However, the addition of three lysines to both termini was found to decrease
hydrophobicity sufficiently to enable HPLC purification. Circular dichroism
studies of a chemically synthesized K3-Ste2p-14-43-K3 peptide indicated
mostly random structure, with ~45% b-strand and a small percentage of a-he-
lix in buffered water. The structure was found to be stable at temperatures up
to 40C. These results correlate with predicted 2o structure for the Ste2p
N-terminal domain including: random chain with a b-strand-loop-b-strand
fold followed by a C-terminal a-helix (Shi et al., J. Cell. Biochem.
107:630-38) and recent NMR evidence suggesting a-helix in a C-terminal
overlapping region (residues 39-47; Neumoin et al., 2009 Biophysical Journal
96: 3187-96). Preliminary 1H-1H NOESY and TOCSY NMR data for theK3-Ste2p 14-43-K3 peptide have been collected. As well, a recombinant
version of K3-Ste2p-2-43-K3 is being produced to extend the N-terminal
region to be analyzed and facilitate isotopic labeling for complete structural
elucidation.
1521-Pos
Arrestin can Bind to a Single G-Protein Coupled Receptor
Hisao Tsukamoto, Abhinav Sinha, Mark DeWitt, David L. Farrens.
Oregon Health and Science University, Portland, OR, USA.
Termination of G protein-coupled receptor (GPCR) signaling typically in-
volves phosphorylation of the receptor, followed by binding of a protein called
arrestin. Here we tested the minimal stoichiometry required for this inter-
action, by determining if a single rhodopsin molecule can bind arrestin. To
do this, we prepared nanoscale phospholipids particles, so-called nanodiscs,
which contain only monomeric rhodopsin and measured their ability to bind
visual arrestin. Our data clearly show that visual arrestin can bind to mono-
meric phosphorylated rhodopsin to stabilize its active form, called metarho-
dopsin II. Interestingly, we find beta-arrestin can also bind to monomeric
rhodopsin in nanodiscs and stabilize metarhodopsin II. Together, these results
suggest that in general, the minimal unit for arrestin binding is a monomeric
GPCR.
1522-Pos
Solid-State NMR Demonstrates that Active Signaling Complexes of Bacte-
rial Chemoreceptors Do Not Adopt the Proposed Trimer-Of-Dimers
Structure
Daniel J. Fowler, Robert M. Weis, Lynmarie K. Thompson.
University of Massachusetts, Amherst, MA, USA.
The receptor dimers that mediate bacterial chemotaxis form signaling com-
plexes with CheW and the kinase CheA. Based on the packing arrangement ob-
served in two different crystal structures of two different receptor cytoplasmic
fragments, two different models have been proposed for receptor signaling
arrays: the trimers-of-dimers and hedgerow models. We have identified an
inter-dimer distance predicted to be substantially different by the two models,
labeled the atoms defining this distance through isotopic enrichment, and mea-
sured it with 19F-13C REDOR. This was done in two types of receptor samples:
isolated bacterial membranes containing overexpressed, intact receptor, and
soluble receptor fragments reconstituted into kinase-active signaling com-
plexes. In both cases, the distance found was incompatible with both the
trimers-of-dimers and the hedgerow models. Comparisons of simulated and
observed REDOR dephasing were used to deduce a closest-approach distance
at this interface, which provides a constraint for the possible arrangements of
receptor assemblies in the kinase-
active signaling state.
This research was supported by
U.S. Public Health Service Grant
GM47601; DJF was partially sup-
ported by National Research Service
Award T32 GM08515.1523-Pos
Site-Specific Fluorescent Labeling of Purified G-Protein-Coupled Recep-
tors Using Genetically-Encoded Unnatural Amino Acids
Shixin Ye, Manija A. Kazmi, Terence Duarte, Saranga Naganathan,
Thomas P. Sakmar, Thomas Huber.
www.sakmarlab.org, Rockefeller University, New York, NY, USA.
The introduction of unique chemical groups into proteins by means of site-
directed mutagenesis with unnatural amino acids has numerous applications
in protein engineering and functional studies. We first introduced p-acetyl-L-
phenylalanine (Acp) or p-azido-L-phenylalanine (Azp) into the prototypical
G protein-coupled receptor (GPCR) rhodopsin at specific sites. We employed
an amber codon suppression system where the mutant opsin gene was co-
expressed with the appropriate
orthogonal pair of engineered tRNA
and amino-acyl tRNA synthetase.
We then used hydrazone (hydrazide)
or Staudinger-Bertozzi (phosphine)
ligation chemistry for the keto group
(in Acp) or azido group (in Azp),
respectively, to link a fluorophore at
various solvent accessible sites in
rhodopsin. In side-by-side compari-
sons of the two chemical ligation
chemistries, which were carried out


Solid-State NMR Demonstrates that Active Signaling Complexes of Bacterial Chemoreceptors Do Not Adopt the Proposed Trimer-Of-Dimers Structure 

file:///data/remote/core/dit/data/elsevier/pdf/735/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUwOTAzMzkyeA==.pdf

Solid-State NMR Demonstrates that Active Signaling Complexes of Bacterial Chemoreceptors Do Not Adopt the Proposed Trimer-Of-Dimers Structure

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector