Christian Dees

Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors

Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling.

Multisite contacts involved in coupling of the β‐adrenergic receptor with the stimulatory guanine‐nucleotide‐binding regulatory protein

Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

Mapping of β-adrenoceptor coupling domains to Gs-protein by site-specific synthetic peptides

Peptides corresponding to the known sequence of turkey erythrocyte β1‐adrenergic receptor were synthesized and the effects on receptor‐mediated cyclase activation were measured. Peptides corresponding to the first and second intracellular loops (T61‐71 and T138‐159) inhibited at micromolar concentrations the hormone‐dependent cyclase activation in turkey erythrocyte membranes. In contrast, the peptide corresponding to the C‐terminal part of the third intracellular loop (T284‐295) increased the cyclase activity in a hormone‐independent manner. Peptides T338‐353 and T2‐10 and a number of synthetic peptides unrelated to the β‐adrenoceptor had no effect.

Identification of a Gs-protein coupling domain to the β-aderenoceptor using site-specific synthetic peptides: Carboxyl terminus of Gsα is involved in coupling to β-adrenoceptors

Competition between Gs‐protein and the synthetic peptide, GSA 379‐394, derived from the carboxyl‐terminal region of the αs‐subunit, led to complete inhibition of receptor‐mediated adenylate cyclase activation in turkey erythrocyte membranes. Related peptides corresponding to the homologous carboxyl‐terminal region of αt‐,αil‐ or αo‐subunits did not interfere with β‐receptor‐Gs coupling. The direct coupling between Gs and adenylate cyclase was not influenced by any of these peptides. These results emphasize the important role of the carboxyl‐terminus of G‐protein α‐subunits for the specific recognition of their corresponding receptors and for signal transduction.

A Fluorescence Resonance Energy Transfer-based M2 Muscarinic Receptor Sensor Reveals Rapid Kinetics of Allosteric Modulation

Allosteric modulators have been identified for several G protein-coupled receptors, most notably muscarinic receptors. To study their mechanism of action, we made use of a recently developed technique to generate fluorescence resonance energy transfer (FRET)-based sensors to monitor G protein-coupled receptor activation. Cyan fluorescent protein was fused to the C terminus of the M2 muscarinic receptor, and a specific binding sequence for the small fluorescent compound fluorescein arsenical hairpin binder, FlAsH, was inserted into the third intracellular loop; the latter site was labeled in intact cells by incubation with FlAsH. We then measured FRET between the donor cyan fluorescent protein and the acceptor FlAsH in intact cells and monitored its changes in real time. Agonists such as acetylcholine and carbachol induced rapid changes in FRET, indicative of agonist-induced conformational changes. Removal of the agonists or addition of an antagonist caused a reversal of this signal with rate constants between 400 and 1100 ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes in FRET when given alone, but increased FRET when given in the presence of an agonist, compatible with an inactivation of the receptors. The kinetics of these effects were very rapid, with rate constants of 80–100 ms and ≈200 ms for saturating concentrations of gallamine and dimethyl-W84, respectively. Because these speeds are significantly faster than the responses to antagonists, these data indicate that gallamine and dimethyl-W84 are allosteric ligands and actively induce a conformation of the M2 receptor with a reduced affinity for its agonists.

Purification and functional characterization of the human β2-adrenergic receptor produced in baculovirus-infected insect cells

A human cDNA fragment bearing the complete coding region for the β2‐adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 104 β2‐adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs‐protein.

Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores.

Identification of a C-terminal binding site for G-protein βγ-subunits in phosducin-like protein

Phosducin‐like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G‐protein βγ‐subunit (Gβγ)‐mediated signaling, with an affinity about 5‐fold lower than that of phosducin. The Gβγ binding site of phosducin has been suggested to be contained in its N‐terminus. A region corresponding to this N‐terminus is lacking in PhLP, suggesting that PhLP must utilize a different mode of Gβγ binding. To map the Gβγ binding site in PhLP, a series of deletion mutants were constructed, expressed in E. coli as glutathione S‐transferase (GST) fusion proteins, and the purified fusion proteins were examined for their ability to attenuate Go GTPase activity. Progressive N‐terminal truncations of PhLP caused only minor reductions in potency, whereas the complementary N‐terminal PhLP fragments turned out to be inactive. We further identified a short C‐terminal segment comprising residues 168 to 195 that inhibited Go GTPase activity similar in efficacy and potency to full‐length PhLP. This C‐terminal fragment was also capable of antagonizing a second Gβγ‐mediated function, the enhancement of rhodopsin phosphorylation by the β‐adrenergic receptor kinase. Taken together, these data indicate that PhLP interacts with Gβγ via a short C‐terminal binding site which is distinct from that identified previously in phosducin.

Live Cell Monitoring of μ-Opioid Receptor-mediated G-protein Activation Reveals Strong Biological Activity of Close Morphine Biosynthetic Precursors

G-protein activation by receptors is generally measured using 35S-GTPγS binding assays in cell membranes and cannot be well assessed in intact cells. We have recently developed a fluorescence resonance energy transfer (FRET)-based approach to monitor Gi-protein activation in living cells. Here we report that this technique can be used to determine structure-activity relationships of receptor agonists in intact cells. We have recently shown that morphine is biosynthesized de novo by mammals via a multistep pathway different from that in plants. However, the pharmacological properties of morphine precursors are poorly understood. Here, we directly monitored μ-opioid receptor (MOR)-mediated Gi-protein activation in living cells by FRET and validated this method with classical GTPγS binding assays. Receptor binding studies and FRET measurements demonstrated that several (R)-configurated morphine precursors such as (R)-reticuline, salutaridine, salutaridinol, thebaine, and codeine were partial MOR agonists. Some closer precursors such as oripavine, codeinone, and morphinone activated Gi-proteins as strongly as morphine, but with slightly lower potencies. The more distant the precursors were positioned in the pathway with respect to morphine, the less efficient and potent they were at MOR. Comparison of pharmacological properties of close morphine precursors and concentrations in which they occur in animal tissues suggests that they might activate MOR signaling under physiological conditions. Taken together, our data indicate that FRET-based assays of G-protein activation can serve to determine the abilities of compounds to activate G-protein signaling directly and in living cells.

G proteins in reverse mode - receptor-mediated GTP release inhibits G protein and effector function

Active G protein-coupled receptors activate heterotrimeric Gαβγ proteins by catalyzing the exchange of GDP by GTP at the Gα subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and Go protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of Go proteins by active α2A-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Gαβγ. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.