Cryo-electron tomography pipeline for plasma membranes.

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes.

Spatial and signaling overlap of growth factor receptor systems at clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these complexes in response to external cues remain unclear. We use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures in human squamous HSC3 cells.

Crosstalk of growth factor receptors at plasma membrane clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these nanoscale complexes in response to external cues remain unclear. Here, we use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures across the plasma membrane of human squamous HSC3 cells.

Dual clathrin and integrin signaling systems regulate growth factor receptor activation.

The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood.

Sites phosphorylated in human α-adrenoceptors in response to noradrenaline and phorbol myristate acetate.

Phosphorylation of the human α-adrenergic receptor (fused with the green fluorescent protein) was studied employing the inducible Flp-ln HEK293 T-Rex system for expression. Serine/alanine substitutions were performed in five sites corresponding to those previously identified as phosphorylation targets in the hamster ortholog. Desensitization was decreased in these mutants but receptor phosphorylation was still clearly detected.

Distinct phosphorylation sites/clusters in the carboxyl terminus regulate α-adrenergic receptor subcellular localization and signaling.

The human α-adrenergic receptor is a seven transmembrane-domain protein that mediates many of the physiological actions of adrenaline and noradrenaline and participates in the development of hypertension and benign prostatic hyperplasia. We recently reported that different phosphorylation patterns control α-adrenergic receptor desensitization. However, to our knowledge, there is no data regarding the role(s) of this receptor's specific phosphorylation residues in its subcellular localization and signaling.

Different phosphorylation patterns regulate α-adrenoceptor signaling and desensitization.

Human α-adrenoceptors (α-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations.

Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α-adrenergic receptors.

In LNCaP cells that stably express α-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization.

Protein Kinase C Activation Promotes α-Adrenoceptor Internalization and Late Endosome Trafficking through Rab9 Interaction. Role in Heterologous Desensitization.

Upon agonist stimulation, -adrenergic receptors couple to G proteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such as -arrestin and clathrin. However, the fine mechanisms that participate remain unsolved.

Novel Structural Approaches to Study GPCR Regulation.

Background: Upon natural agonist or pharmacological stimulation, G protein-coupled receptors (GPCRs) are subjected to posttranslational modifications, such as phosphorylation and ubiquitination. These posttranslational modifications allow protein-protein interactions that turn off and/or switch receptor signaling as well as trigger receptor internalization, recycling or degradation, among other responses. Characterization of these processes is essential to unravel the function and regulation of GPCR.

Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway.

Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states.

α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion.