Rapid Activation of Inwardly Rectifying Potassium Channels by Immobile G-Protein-Coupled Receptors

Lober, Robert M.; Pereira, Miguel Alvares; Lambert, Nevin A.

doi:10.1523/jneurosci.4020-06.2006

Cited by 33 publications

(22 citation statements)

References 38 publications

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“…We have previously shown that similar fluorescent protein-mOR fusion proteins are functional when immobilized on the cell surface (Lober et al, 2006). Accordingly, agonist stimulation (10 mM DAMGO) rapidly redistributed V-arrestin3 from the cytosol to the bead-associated plasma membrane in cells expressing His-C-mOR (Supplemental Fig.…”

Section: Resultsmentioning

confidence: 93%

Segregation of Family A G Protein–Coupled Receptor Protomers in the Plasma Membrane

et al. 2013

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G protein-coupled receptors (GPCRs) transduce many important physiological signals and are targets for a large fraction of therapeutic drugs. Members of the largest family of GPCRs (family A) are thought to self-associate as dimers and higher-order oligomers, although the significance of such quaternary structures for signaling or receptor trafficking is known for only a few examples. One outstanding question is the physical stability of family A oligomers in cell membranes. Stable oligomers would be expected to move through cellular compartments and membrane domains as intact groups of protomers. Here, we test this prediction by recruiting subsets of affinity-tagged family A protomers into artificial microdomains on the surface of living cells and asking if untagged protomers move into these domains (are corecruited) at the same time. We find that tagged b 2 adrenergic and m-opioid protomers are unable to corecruit untagged protomers into microdomains. In contrast, tagged metabotropic glutamate receptor protomers do corecruit untagged protomers into such microdomains, which is consistent with the known covalent mechanism whereby these family C receptors dimerize. These observations suggest that interactions between these family A protomers are too weak to directly influence subcellular location, and that mechanisms that move these receptors between subcellular compartments and domains must operate on individual protomers.

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Section: Resultsmentioning

confidence: 93%

Segregation of Family A G Protein–Coupled Receptor Protomers in the Plasma Membrane

et al. 2013

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“…Yet, despite this highly transient nature, the FRAP of ␤ 1 ␥ 2 -activated PLC␤ 2 (or PLC␤ 2 ⌬) is strongly dominated by fast lateral diffusion (Figs. 4 and 5), characterized by D values 3-to 5-fold faster than lipids or lipidanchored proteins such as G protein ␣ or ␤␥ subunits (7,32,34,62,63). Combining the highly transient interactions and the very fast diffusion along the plasma membrane, we propose that stimulation by ␤ 1 ␥ 2 induces surfing-like diffusion of PLC␤ 2 enzymes along the cytoplasmic face of the plasma membrane.…”

Section: Discussionmentioning

confidence: 91%

“…Nevertheless, the association of ␣ q -stimulated PLC␤ 2 with membrane constituents (including ␣ q itself) must be dynamic, as indicated by the contribution of exchange to the FRAP kinetics (Fig. 4) and by the lower D value (0.47 m 2 /s) measured for a G␣ subunit (␣ oA ) (63). Based on the D and ex values, the travel range of ␣ q -stimulated PLC␤ 2 is estimated to be 0.75 m, suggesting that PLC␤ 2 recruited by ␣ q stimulation is less constrained than the Rac2-stimulated enzyme, roaming larger (albeit still limited) membrane regions.…”

Section: Discussionmentioning

confidence: 99%

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Gutman

Walliser

Piechulek

et al. 2010

Journal of Biological Chemistry

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We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-␤ 2 (PLC␤ 2 ) by G protein ␣ q and ␤␥ dimers. We show that activation by ␣ q and ␤␥ differ from activation by Rac2 and from each other. Stimulation by ␣ q enhanced the plasma membrane association of PLC␤ 2 , but not of PLC␤ 2 ⌬, which lacks the ␣ q -interacting region. Although ␣ q resembled Rac2 in increasing the contribution of exchange to the FRAP of PLC␤ 2 and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLC␤ 2 by ␣ q occurred by enhancing PLC␤ 2 association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by ␤␥ shifted the FRAP of PLC␤ 2 and PLC␤ 2 ⌬ to pure lateral diffusion 3-to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLC␤ 2 membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) in localized versus dispersed populations. PLC␤ 2 activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLC␤ 2 to act locally on PtdInsP 2 at specific domains. Activation by ␣ q leads to lipid-like diffusion of PLC␤ 2 accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by ␤␥ recruits PLC␤ 2 to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP 2 populations. Phospholipase C-␤ (PLC␤)4 isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) to produce inositol 1,4,5-trisphosphate and diacylglycerol (1-3). They are activated to different extents by heterotrimeric G protein ␣ q subunits (␣ q ) and ␤␥ dimers (␤␥) (1-3). PLC␤ 2 is also activated by the Rho GTPases Rac and Cdc42 (4 -7). Activation by Rac has also been demonstrated for PLC␥ 2 (8). PLC␤ 2 has long been known to be expressed in hematopoietic cells (2, 3) but is also encountered in a variety of other cell types and tissues, including smooth muscle cells (9) and several brain regions (10, 11). Moreover, PLC␤ 2 was shown to be essential for taste perception via certain G protein-coupled oral taste receptors (12).Activation of PLC␤ 2 by ␣ q and related ␣ subunits requires the C-terminal region of the enzyme; mutants with deletions in this region (e.g. PLC␤ 2 ⌬, that lacks the Phe 819 -Glu 1166 segment) are resistant to stimulation by ␣ q , but undergo activation by ␤␥ and Rac/Cdc42 (7, 13-15). Recent results show that ␤␥ and Rac/Cdc42 activate PLC␤ 2 by interacting, at least in part, with different regions of the effector enzyme. Thus, although the pleckstrin homology (PH) domain of PLC␤ 2 is dispensable for activation of the enzyme by...

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“…Where functional studies have been done using the activation of GIRK-dependent currents, precoupling has been suggested (Zhang et al, 2002;Lober et al, 2006;Zhou et al, 2012). Those experiments examined receptors (including MOR) that were immobilized, and the mobility of G proteins, GIRK channels, or RGS proteins was assayed (Zhang et al, 2002;Lober et al, 2006;Zhou et al, 2012).…”

Section: Functional Mors After Desensitization Discussionmentioning

confidence: 99%

Desensitization of Functionalµ-Opioid Receptors Increases Agonist Off-Rate

Williams

2014

Mol Pharmacol

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Desensitization of m-opioid receptors (MORs) develops over 5-15 minutes after the application of some, but not all, opioid agonists and lasts for tens of minutes after agonist removal. The decrease in function is receptor selective (homologous) and could result from 1) a reduction in receptor number or 2) a decrease in receptor coupling. The present investigation used photolysis of two caged opioid ligands to examine the kinetics of MOR-induced potassium conductance before and after MOR desensitization. Photolysis of a caged antagonist, carboxynitroveratrylnaloxone (caged naloxone), blocked the current induced by a series of agonists, and the time constant of decline was significantly decreased after desensitization. The increase in the rate of current decay was not observed after partial blockade of receptors with the irreversible antagonist, b-chlornaltrexamine (b-CNA). The time constant of current decay after desensitization was never more rapid than 1 second, suggesting an increased agonist off-rate rather than an increase in the rate of channel closure downstream of the receptor. The rate of G proteincoupled K 1 channel (GIRK) current activation was examined using photolysis of a caged agonist, carboxynitrobenzyl-tyrosine- [Leu 5 ]-enkephalin. After acute desensitization or partial irreversible block of MORs with b-CNA, there was an increase in the time it took to reach a peak current. The decrease in the rate of agonist-induced GIRK conductance was receptor selective and dependent on receptor number. The results indicate that opioid receptor desensitization reduced the number of functional receptor and that the remaining active receptors have a reduced agonist affinity.

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Rapid Activation of Inwardly Rectifying Potassium Channels by Immobile G-Protein-Coupled Receptors

Cited by 33 publications

References 38 publications

Segregation of Family A G Protein–Coupled Receptor Protomers in the Plasma Membrane

Segregation of Family A G Protein–Coupled Receptor Protomers in the Plasma Membrane

Differential Regulation of Phospholipase C-β2 Activity and Membrane Interaction by Gαq, Gβ1γ2, and Rac2

Desensitization of Functionalµ-Opioid Receptors Increases Agonist Off-Rate

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