high-resolution-spatiotemporal-analysis-receptor-dynamics-single

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

University of Würzburg, Germany

Oxidative stress and hyper-functioning of angiotensin II receptor type I (AT(1)R) are commonly observed in hypertensive patients but the relationship between oxidative stress and AT(1)R function is still unclear. We investigated the effects of H(2)O(2) treatment on AT(1)R-mediated intracellular calcium [Ca(2+)](i) signaling and its cell surface distribution pattern in HEK cells stably expressing EGFP-tagged rat AT(1)R using image correlation spectroscopy (ICS). Following H(2)O(2) treatment (50-800μM), [Ca(2+)](i) was significantly increased upon angiotensin II stimulation. Similarly ICS revealed a significant increase in degree of AT(1)R aggregation in H(2)O(2) treated group during Ang II activation but no difference in cluster density compared with untreated control cells or those with N-acetyl cysteine pretreatment. Thus, oxidative stress-induced AT(1)R hyper-responsiveness can be attributed by an increase in cell surface receptor aggregation state, possibly stemming in part from oxidant-related increase receptor-receptor interactions.

Oxidative stress increases angiotensin receptor type I responsiveness by increasing receptor degree of aggregation using image correlation spectroscopy.

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.

Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.

Angiotensin II receptor type I (AT1R) is a G-protein coupled receptor involved in regulation of body water-electrolyte balance and blood pressure. Oxidative stress promotes AT1R oligomerization and hyper-responsiveness to its cognate ligand Ang II. In this study, bivalent Ang II, synthesized by linking with aminocaproic acid (Acp) at the N-terminus, was used to induce AT1R dimerization and hyper-responsiveness in AT1R-expressed human embryonic kidney (AT1R-HEK) cells, determined using image correlation spectroscopy (ICS) and by measuring AT1R-mediated change in intracellular Ca(2+) concentration, respectively. In addition, ICS was employed to determine distribution pattern of cell-surface AT1R and its degree of aggregation when stimulated by monomeric (monovalent) and bivalent Ang II under oxidative stress (100 μM H2O2) condition in comparison with normal (unoxidized) AT1R-HEK cells. Bivalent Ang II induced cell-surface AT1R aggregation/clustering but maintained AT1R normal signaling response under oxidative stress condition, whereas stimulation by monomeric Ang II or a mixture of monomeric and Acp-modified Ang II (used in the synthesis of bivalent form) resulted in AT1R hyper-responsiveness. These results suggest that bivalent ligand (viz. Ang II) provides another strategy in the development of novel drugs specifically designed for attenuating aberrant responsiveness of cognate receptor (AT1R) under pathological (oxidative stress) conditions.

Titiwat Sungkaworn

Institute of Pharmacology and Toxicology and Bio-Imaging Center/Rudolf Virchow Center,
DFG-Research Center for Experimental Biomedicine,
Institute of Pharmacology and Toxicology and Bio-Imaging Center/Rudolf Virchow Center, DFG-Research Center for Experimental Biomedicine

University of Würzburg, Germany

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy