20 research outputs found
Agonists activate different A2B adenosine receptor signaling pathways in MBA-MD-231 breast cancer cells with distinct potencies
Chemogenetic and Optogenetic Activation of Gαs Signaling in the Basolateral Amygdala Induces Acute and Social Anxiety-Like States
Sex hormone-related neurosteroids differentially rescue bioenergetic deficits induced by amyloid-β or hyperphosphorylated tau protein
Optical functionalization of human Class A orphan G-protein-coupled receptors
G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca2+-, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33, we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs.</p
Optogenetic stimulation of medial prefrontal cortex Drd1 neurons produces rapid and long-lasting antidepressant effects
Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures
Optical regulation of class C GPCRs by photoswitchable orthogonal remotely tethered ligands
G protein-coupled receptors (GPCRs) respond to a wide range of extracellular cues to initiate complex downstream signaling cascades that control myriad aspects of cell function. Despite a long-standing appreciation of their importance to both basic physiology and disease treatment, it remains a major challenge to understand the dynamic activation patterns of GPCRs and the mechanisms by which they modulate biological processes at the molecular, cellular, and tissue levels. Unfortunately, classical methods of pharmacology and genetic knockout are often unable to provide the requisite precision needed to probe such questions. This is an especially pressing challenge for the class C GPCR family which includes receptors for the major excitatory and inhibitory neurotransmitters, glutamate and GABA, which signal in a rapid, spatially-delimited manner and contain many different subtypes whose roles are difficult to disentangle. The desire to manipulate class C GPCRs with spatiotemporal precision, genetic targeting, and subtype specificity has led to the development of a variety of photopharmacological tools. Of particular promise are the photoswitchable orthogonal remotely tethered ligands (“PORTLs”) which attach to self-labeling tags that are genetically encoded into full length, wild-type metabotropic glutamate receptors (mGluRs) and allow the receptor to be liganded and un-liganded in response to different wavelengths of illumination. While powerful for studying class C GPCRs, a number of detailed considerations must be made when working with these tools. The protocol included here should provide a basis for the development, characterization, optimization, and application of PORTLs for a wide range of GPCRs