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.
G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β 1 -adrenergic receptor (β 1 AR), the β 2 -adrenergic receptor (β 2 AR), and the γ-aminobutyric acid (GABA B ) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β 1 ARs (monomers/dimers) and highest for GABA B receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β 1 -/ β 2 ARs were apparently freely diffusing on the cell surface, GABA B receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA B receptor complexes. These data provide a spatiotemporal characterization of β 1 -/β 2 ARs and GABA B receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.live cell imaging | protein-protein interactions
BACKGROUND
Corticotropin-independent Cushing’s syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood.
METHODS
We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro.
RESULTS
Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP–dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing’s syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased.
CONCLUSIONS
Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.)
G-protein-coupled receptors mediate the biological effects of many hormones and neurotransmitters and are important pharmacological targets. They transmit their signals to the cell interior by interacting with G proteins. However, it is unclear how receptors and G proteins meet, interact and couple. Here we analyse the concerted motion of G-protein-coupled receptors and G proteins on the plasma membrane and provide a quantitative model that reveals the key factors that underlie the high spatiotemporal complexity of their interactions. Using two-colour, single-molecule imaging we visualize interactions between individual receptors and G proteins at the surface of living cells. Under basal conditions, receptors and G proteins form activity-dependent complexes that last for around one second. Agonists specifically regulate the kinetics of receptor-G protein interactions, mainly by increasing their association rate. We find hot spots on the plasma membrane, at least partially defined by the cytoskeleton and clathrin-coated pits, in which receptors and G proteins are confined and preferentially couple. Imaging with the nanobody Nb37 suggests that signalling by G-protein-coupled receptors occurs preferentially at these hot spots. These findings shed new light on the dynamic interactions that control G-protein-coupled receptor signalling.
The use of low b-TSH cutoff allowed the detection of an unsuspected number of children with neonatal hypothyroidism, evolving in mild permanent thyroid dysfunction later in life. The incidence of CH in this Italian population appears to be double than previously thought with a clear-cut prevalence of functional defects over dysgenetic ones.
Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3-6 d.
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
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