scholarly journals Structural insights into differences in G protein activation by family A and family B GPCRs

Science ◽  
2020 ◽  
Vol 369 (6503) ◽  
pp. eaba3373 ◽  
Author(s):  
Daniel Hilger ◽  
Kaavya Krishna Kumar ◽  
Hongli Hu ◽  
Mie Fabricius Pedersen ◽  
Evan S. O’Brien ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Complexes ◽  
Structural Difference ◽  
Guanine Nucleotide Binding Protein ◽  
Glucagon Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Protein Dissociation ◽  
Α Helix ◽  
Double Electron Electron Resonance

Family B heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β2 adrenergic receptor (β2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo–electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the β2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

scholarly journals Crystal structure of rhodopsin in complex with a mini-Gosheds light on the principles of G protein selectivity

2018 ◽  
Vol 4 (9) ◽  
pp. eaat7052 ◽  
Author(s):  
Ching-Ju Tsai ◽  
Filip Pamula ◽  
Rony Nehmé ◽  
Jonas Mühle ◽  
Tobias Weinert ◽  
...  
Keyword(s):  
Crystal Structure ◽  
G Protein ◽  
Maximum Efficiency ◽  
Guanine Nucleotide Binding Protein ◽  
Precise Position ◽  
Protein Activation ◽  
Signaling Complex ◽  
G Protein Activation ◽  
Clinical Drugs ◽  
Guanine Nucleotide Binding

Selective coupling of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-Goprotein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a “structural switch,” allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal “hook-like” element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation.

scholarly journals Dual Lipid Modification Motifs in Gαand GγSubunits Are Required for Full Activity of the Pheromone Response Pathway inSaccharomyces cerevisiae

2000 ◽  
Vol 11 (3) ◽  
pp. 957-968 ◽  
Author(s):  
Carol L. Manahan ◽  
Madhavi Patnana ◽  
Kendall J. Blumer ◽  
Maurine E. Linder
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Yeast Cells ◽  
Pheromone Response ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Protein Activation ◽  
Pheromone Response Pathway ◽  
G Protein Activation

To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide–binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein γ subunit (Ste18p) is unusual among Gγsubunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the Gγsubunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of Gβγafter receptor-stimulated release from Gα. The G protein α subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

scholarly journals Heterogeneity of G-protein activation by the calcium-sensing receptor

2021 ◽  
Author(s):  
Hasnat Ali Abid ◽  
Asuka Inoue ◽  
Caroline M. Gorvin
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Receptor Activation ◽  
Assay System ◽  
Hek293 Cells ◽  
Calcium Sensing Receptor ◽  
Protein Activation ◽  
Calcium Sensing ◽  
G Protein Activation ◽  
Protein Dissociation

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G-protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messenger proteins or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G-protein activation directly, we took advantage of a recently described NanoBiT G-protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G-proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G-proteins were similarly expressed, suggesting CaSR may activate multiple G-proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.

Opioid-Activated Postsynaptic, Inward Rectifying Potassium Currents in Whole Cell Recordings in Substantia Gelatinosa Neurons

1998 ◽  
Vol 80 (6) ◽  
pp. 2954-2962 ◽  
Author(s):  
S. P. Schneider ◽  
W. A. Eckert ◽  
A. R. Light
Keyword(s):  
Membrane Potential ◽  
G Protein ◽  
Potassium Currents ◽  
Substantia Gelatinosa ◽  
Opioid Agonist ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Protein Activation ◽  
G Protein Activation ◽  
Whole Cell Recordings

Schneider, S. P., W. A. Eckert III, and A. R. Light. Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954–2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the μ-opioid agonist (d-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1–5 μM DAMGO caused a robust and repeatable hyperpolarization in membrane potential ( V m) and decrease in neuronal input resistance ( R N) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5′-triphosphate (GTP; 100 μM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5′- O-(3-thiotriphosphate) (GTP-γ-S; 100 μM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5′- O-(2-thiodiphosphate) (GDP-β-S; 500 μM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate μ-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

scholarly journals β-Amyloid Peptide-induced Apoptosis Regulated by a Novel Protein Containing a G Protein Activation Module

2001 ◽  
Vol 276 (22) ◽  
pp. 18748-18756 ◽  
Author(s):  
Eileen M. Kajkowski ◽  
C. Frederick Lo ◽  
Xiaoping Ning ◽  
Stephen Walker ◽  
Heidi J. Sofia ◽  
...  
Keyword(s):  
G Protein ◽  
Amyloid Peptide ◽  
Protein Activation ◽  
G Protein Activation ◽  
Β Amyloid ◽  
Β Amyloid Peptide ◽  
Induced Apoptosis ◽  
Novel Protein
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