G-protein-coupled receptor kinases and arrestins: regulators of G-protein-coupled receptor sequestration.
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Animals↗
Biomedical subjects
S S Ferguson
Publications and source records attributed to S S Ferguson.
G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins.
G-protein-coupled receptors (GPCRs) represent a large family of proteins that transduce extracellular signals to the interior of cells. Signalling through these receptors rapidly desensitized primarily as the consequence of receptor phosphorylation, but receptor sequestration and downregulation can also contribute to this process. Two families of serine/threonine kinases, second messenger dependent protein kinases and receptor-specific G-protein-coupled receptor kinases (GRKs), phosphorylate GPCRs and thereby contribute to receptor desensitization. Receptor-specific phosphorylation of GPCRs promotes the binding of cytosolic proteins referred to as arrestins, which function to further uncouple GPCRs from their heterotrimeric G-proteins. To date, the GRK protein family consists of six members, which can be further classified into subgroups according to sequence homology and functional similarities. The arrestin protein family also comprises six members, which are subgrouped on the basis of sequence homology and tissue distribution. While the molecular mechanisms contributing to GPCR desensitization are fairly well characterized, little is known about the mechanism(s) by which GPCR responsiveness is reestablished, other than that receptor sequestration (internalization) might be involved. The goal of the present review is to overview current understanding of the regulation of GPCR responsiveness. In particular, we will review new evidence suggesting a pleiotropic role for GRKs and arrestins in the regulation of GPCR responsiveness. GRK-mediated phosphorylation and arrestin binding are not only involved in the functional uncoupling of GPCRs but they are also intimately involved in promoting GPCR sequestration and as such likely play an important role in mediating the subsequent resensitization of GPCRs.
Animals↗
The conserved seven-transmembrane sequence NP(X)2,3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor.
The beta 2-adrenergic receptor (beta 2AR) is a member of a large superfamily of seven transmembrane domain, G-protein-coupled receptors. Within the putative seventh transmembrane domain of the beta 2AR is a sequence of amino acids, NPLIY, which is conserved with minor variations in all members of the superfamily. Previously it was demonstrated that mutation of tyrosine residue 326 to an alanine abolished agonist promoted sequestration of this mutant without affecting its ability to maximally stimulate adenylyl cyclase in membranes [Barak, L.S., Tiberi, M., Freedman, N.J., Kwatra, M.M., Lefkowitz, R.J., & Caron M.J. (1994) J Biol. Chem. 269, 2790-2795]. In the present study we characterized the NPLIY amino acid sequence in an attempt to determine how it can affect the agonist-mediated sequestration of the beta 2AR and to test whether it is a functional motif. We find that point mutations of the most conserved amino acids, N, P, and Y, in this sequence affect several other receptor properties in addition to sequestration. Mutation of asparagine 322 to an alanine resulted in complete uncoupling of the receptor, loss of high-affinity agonist binding, and abolition of receptor sequestration, down-regulation, and phosphorylation. In contrast, a conservative mutation of this residue to an aspartic acid (as found in the thrombin receptor) resulted in an improvement of G-protein coupling without adversely affecting other receptor properties. Substitution of proline residue 323 with an alanine residue resulted in a receptor with mild deficits in sequestration and coupling, a reduced agonist-mediated phosphorylation, and no change in down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.
The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.
Adrenergic beta-Agonists↗
Stereoselectivity of the inhibition of [3H]hemicholinium-3 binding to the sodium-dependent high-affinity choline transporter by the enantiomers of alpha- and beta-methylcholine.
In a previous report, we showed that the enantiomers of alpha- and beta-methylcholine inhibited choline uptake with stereoselectivity, but that their transport by the choline carrier of nerve terminals showed stereospecificity. The present experiments used the same choline analogues to determine if either of the above characteristics pertains to their ability to interact with the [3H]-hemicholinium-3 binding site present on striatal membranes and synaptosomes. [3H]Hemicholinium-3 binding to striatal membranes could be inhibited stereoselectively by the enantiomers of beta-methylcholine, but R(+)-alpha-methylcholine was little better than its enantiomer in this test. However, [3H]hemicholinium-3 binding to striatal synaptosomes was inhibited stereoselectively by the enantiomers of both alpha- and beta-methylcholine. This difference between the properties of [3H]hemicholinium-3 binding to membranes or to synaptosomes appears related to the presence of two ligand binding states. The [3H]hemicholinium-3 binding site could be shifted to a low-affinity state by ATP treatment and to a high-affinity state by EDTA washing. When the [3H]hemicholinium-3 binding site existed in its low-affinity state, binding was inhibited stereoselectively by the enantiomers of both alpha- and beta-methylcholine, but when shifted to its high-affinity state, it was inhibited stereoselectively only by the enantiomers of beta-methylcholine. We conclude that hemicholinium-3 interacts with the substrate recognition site of the high-affinity choline transporter, but that the stereoselectivity of this site changes depending on its affinity state.
Animals↗
Regulation of rat brain synaptosomal [3H]hemicholinium-3 binding and [3H]choline transport sites following exposure to choline mustard aziridinium ion.
Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the "ground-state" (basal) high-affinity choline transport but only 50% of "ground-state" hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K(+)-stimulated increases in choline transport and of [3H]-hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [3H]-hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca2+ or presence of high Mg2+ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg2+ levels removed the ability of the mustard to inhibit K(+)-stimulated increases of either [3H]-hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [3H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extra-cellular Ca2+.
Animals↗
Anaesthetists, nurses and pre-operative visiting.
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Anesthesiology↗
Attention modulates somatosensory cerebral blood flow response to vibrotactile stimulation as measured by positron emission tomography.
In human primary somatosensory cortex, the cerebral blood flow response to vibrotactile stimulation of the fingers (110 Hz), as measured by positron emission tomography and H2(15)O, was 13% higher (p less than 0.025) when the subjects attended to the stimulus, compared to when they were simultaneously engaged in a distraction task. This suggests that the physiological response of a primary cortical area can be modulated by the attentive behavior of the subject.
Adult↗
Stereospecificity of high- and low-affinity transport of choline analogues into rat cortical synaptosomes.
The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)-alpha-methylcholine and S(+)-beta-methylcholine were significantly better inhibitors of the high-affinity choline transport system than were their enantiomers. Although both enantiomers of alpha- and of beta-methylcholine inhibited [3H]choline transport, only R(+)-alpha-methylcholine and S(+)-beta-methylcholine could be transported by the high-affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high-affinity transporter are clearly different. In addition to high-affinity choline transport, Na(+)-independent low-affinity transport was measured. This process transported R(+)-alpha-methylcholine, but not S(-)-alpha-methylcholine; however, it showed no stereoselectivity for the enantiomers of beta-methylcholine. Thus, high- and low-affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.
Animals↗
Agonist-specific regulation of delta-opioid receptor trafficking by G protein-coupled receptor kinase and beta-arrestin.
Opioid receptors mediate multiple biological functions through their interaction with endogenous opioid peptides as well as opioid alkaloids including morphine and etorphine. Previously we have reported that the ability of distinct opioid agonists to differentially regulate mu-opioid receptor (mu OR) responsiveness is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the receptor (1). In the present study, we further examined the role of GRK and beta-arrestin in agonist-specific regulation of the delta-opioid receptor (delta OR). While both etorphine and morphine effectively activate the delta OR, only etorphine triggers robust delta OR phosphorylation followed by plasma membrane translocation of beta-arrestin and receptor internalization. In contrast, morphine is unable to either elicit delta OR phosphorylation or stimulate beta-arrestin translocation, correlating with its inability to cause delta OR internalization. Unlike for the mu OR, overexpression of GRK2 results in neither the enhancement of delta OR sequestration nor the rescue of delta OR-mediated beta-arrestin translocation. Therefore, our findings not only point to the existence of marked differences in the ability of different opioid agonists to promote delta OR phosphorylation by GRK and binding to beta-arrestin, but also demonstrate differences in the regulation of two opioid receptor subtypes. These observations may have important implications for our understanding of the distinct ability of various opioids in inducing opioid tolerance and addiction.
Amino Acid Sequence↗