Research reportG protein-coupled receptor kinases, β-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution
Introduction
The regulation of heptahelical G protein-coupled receptor responsiveness is a relevant issue in neurobiology, and particularly in neuropsychiatric disorders because most psychotropic drugs target one of these receptors. Neurotransmitter receptors are regulated in a complex manner, which can finally lead to receptor desensitization and down-regulation [36]. One important mechanism for this process is the uncoupling of the activated receptors from further stimulation of their G proteins. This form of homologous desensitization is mediated by the phosphorylation of the activated receptor by G protein-coupled receptor kinases (GRKs) [21], [30]. GRK-phosphorylated receptors (on serine/threonine residues at the intracellular C-terminal segment) bind to an arrestin protein (β-arrestin 2/3), which prevents the receptor from activating more G proteins despite the continued binding of agonist [21], [30]. This process is frequently followed by clathrin-coated pit internalization of the arrestin-bound receptors [18], [21], [23]. Following endocytosis, receptors can be dephosphorylated by membrane-associated phosphatases (e.g. type protein phosphatase-2A, PP-2A, a multimeric serine/threonine phosphatase) [25], [29], which eventually results in receptor resensitization and/or a change in receptor signaling [22], [24].
Seven distinct GRKs are known, which can be classified into three distinct groups based on gene structure, sequence similarity, function, and regulation: GRK1-like (GRKs 1 and 7, also named opsin or rhodopsin kinases), GRK2-like (GRKs 2 and 3, formerly named β-adrenergic receptor kinase, β-ARK1/2), and GRK4-like (GRKs 4, 5 and 6) [21], [30], [31]. GRK2/3 possess a pleckstrin-homology-domain (relevant for protein kinase C binding) and are regulated by G protein βγ-subunits; by contrast GRK4/5/6 are insensitive to regulation by Gβγ-subunits [30]. The five extra-retinal GRKs (GRKs 2–6) are expressed widely and abundantly (mRNA and/or protein) in brain and other tissues [2], [31] and GRK-2-like enzymes are the most thoroughly investigated. Initial phenotypical analysis of mutant mice harboring disruptions of specific GRKs or β-arrestins has begun to shed light on the physiological roles of the kinases. Thus, heterozygous GRK2 knockout mice display enhanced adrenergic sensitivity in heart suggesting that β-adrenoceptors are targets of this kinase [38]. On the other hand, functional deletion of the β-arrestin-2 gene in mice results in remarkable potentiation and prolongation of morphine analgesia [5] and also in blockade of opiate tolerance but not dependence [6], which provided clear evidence in vivo for the physiological importance of β-arrestin-2 in regulating the function of a specific G protein-coupled receptor.
Little is known on the possible involvement of brain GRKs and associated regulatory proteins in neuropsychiatric disorders [13], [26]. It is known, however, that the responsiveness of many neurotransmitter receptors is altered during aging, as a result of age-related reductions in receptor density [1], [32], [33] or alterations in signal transduction pathways [32], [33], [39]. At present, the use of postmortem brain tissue represents the only approach that allows an analysis of the pathophysiological conditions of human brain at the level of receptor regulation by GRKs and associated proteins. However, postmortem human brain studies present several methodological issues that need to be addressed in carefully designed studies, and two of the most important variables are the unavoidable delays between death and tissue dissection and the age of subjects, both of which could result in marked postmortem changes and age-related modulations of neurotransmitter receptor regulatory proteins [3], [19], [28].
In this context, the aim of this study was to quantitate in a large number of human brains the immunodensities of two representative GRKs (i.e. GRK2 and GRK6) and those of β-arrestin-2 and other associated regulatory proteins (Gβ protein and PP-2A) to assess, among other methodological issues, for postmortem changes and to investigate the effect of normal aging on these neurotransmitter receptor regulatory mechanisms. Also, the subcellular distribution (cytosol and membrane fractions) of these regulatory proteins was assessed in the human brain. Thus, the main goal was to clarify the influence of a number of crucial parameters that might impact intracellular effectors of signal transduction when using postmortem human brain samples.
Section snippets
Subject selection
Human brains were obtained at autopsy from the Institute of Forensic Medicine, University of Geneva, Switzerland. In typical conditions, the corpse is stored at refrigeration temperature (4 °C) until autopsy. Samples from the right prefrontal cortex (Brodmann’s area 9) were dissected at the time of autopsy and immediately stored at −80 °C until assay. To assess for postmortem changes and the effect of normal aging on GRKs and associated regulatory proteins, brain specimens were taken from
Characterization of antibodies for immunoblot detection of G protein-coupled receptor kinases (GRK2 and GRK6) and β-arrestin-2 in the human brain
Some of the antibodies used were first tested for their specificity on Western blots of human brain tissue. Immunoblot analysis of the prefrontal cortex with anti-GRK2 antibody demonstrated the presence of two immunoreactive bands with molecular masses of ∼80 kDa and ∼74 kDa (Fig. 1 top), in general agreement with previous findings with a different anti-GRK2 antiserum [13], [26]. The higher molecular mass peptide is in the range described for GRK2 protein, whereas the lower band has been
Discussion
The regulation of G protein-coupled receptors is mediated by a complex set of highly conserved molecular mechanisms [9], [21], [36]. One of the most relevant mechanisms for neurotransmitter receptor regulation (homologous regulation) is that induced by specific GRKs leading to receptor phosphorylation, this process is followed by membrane recruitment of β-arrestins which then link receptors to the clathrin-coated pit endocytic process [21], [30]. In general GRKs preferentially phosphorylate
Acknowledgements
This study was supported by grants 31-52242.97, 32-57066.99 and 31-66639.01 from Fonds National Suisse de la Recherche Scientifique (FNSRS, Bern, Switzerland). M. Grange-Midroit was supported by a postdoctoral fellowship and M. Ferrer-Alcón by a predoctoral fellowship from the FNSRS. J.A. Garcı́a-Sevilla is a member of the Institut d’Estudis Catalans (Barcelona, Spain).
References (39)
- et al.
Effect of aging in human cortical pre- and postsynaptic serotonin binding sites
Brain Res.
(1993) - et al.
The ins and outs of intermediate filament organization
Trends Cell. Biol.
(2000) - et al.
Postmortem changes in the level of brain proteins
Exp. Neurol.
(2001) - et al.
The G-protein beta/gamma complex
Cell. Signal.
(1998) - et al.
Heterologous inhibition of G protein-coupled receptor endocytosis mediated by receptor-specific trafficking of β-arrestins
J. Biol. Chem.
(2001) G protein-coupled receptors. New roles for receptor kinases and β-arrestins in receptor signaling and desensitization
J. Biol. Chem.
(1998)- et al.
G protein-coupled receptor kinases
Kidney Int.
(1996) - et al.
G-protein-coupled receptors and signaling networks: emerging paradigms
Trends Pharmacol. Sci.
(2001) - et al.
Regulation of protein kinase cascades by protein phosphatase 2A
Trends Biochem. Sci.
(1999) - et al.
Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway
J. Biol. Chem.
(1998)
Human brain neurochemistry. Some postmortem problems
Life Sci.
The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization and sequence conservation
J. Biol. Chem.
Relationships between β- and α2-adrenoceptors and G coupling proteins in the human brain: effects of age and suicide
Brain Res.
Dynamic complexes of β2-adrenergic receptors with protein kinases and phosphatases and the role of gravin
J. Biol. Chem.
Downregulation of G protein-coupled receptors
Curr. Opin. Neurobiol.
Physiological roles of G-protein-coupled receptor kinases revealed by gene-targeting technology
Trends Pharmacol. Sci.
The G protein-coupled receptor kinase beta ARK1 and beta ARK2 are widely distributed at synapses in the rat brain
J. Neurosci.
Neuropathology of suicide. A review and approach
Ann. NY Acad. Sci.
Age-related changes in frontal and temporal lobe volumes in men
Arch. Gen. Psychiatry
Cited by (29)
Age-dependent antinociception and behavioral inhibition by morphine
2018, Pharmacology Biochemistry and BehaviorCitation Excerpt :Older patients with impaired cytochrome P450 (CYP) enzyme activity and/or renal impairment, are known to be susceptible to opioid toxicity (Gasche et al., 2004; Solus et al., 2004; Klotz, 2009; Eissing et al., 2012; Hocum et al., 2016). Similarly, G protein-coupled receptor kinases (GRK2, GRK6) and β-arrestin expression decrease in an age-dependent manner (Grange-Midroit et al., 2002). Repeated opioid exposure leads to inactivation of opioid receptors by G protein–coupled receptor kinases (GRKs) by phosphorylation and subsequent β-arrestin binding, which reduces the efficacy of opioids (Ferguson et al., 1996; Zhao et al., 2012; Birdsong et al., 2015).
Dysregulation of cannabinoid CB<inf>1</inf> receptor and associated signaling networks in brains of cocaine addicts and cocaine-treated rodents
2013, NeuroscienceCitation Excerpt :The subjects had minimal agonal states with little if any repercussions on brain tissue quality (Preece and Cairns, 2003; García-Fuster et al., 2008a; Álvaro-Bartolomé et al., 2011). The postmortem interval (PMI) is the most important confounding variable in this type of study (Grange-Midroit et al., 2002; Stan et al., 2006). To assess the effect of PMI on CB receptors and neuron-specific enolase (NSE-2) in the PFC/BA9, an independent series of 12 healthy men (negative drug blood toxicology) with different PMIs (range 5–79 h) was used.
Tissue dependent differences in G-protein coupled receptor kinases associated with 5-HT<inf>4</inf> receptor desensitization in the rat gastro-intestinal tract
2011, Biochemical PharmacologyCitation Excerpt :However, the anti-GRK3 antibody labels both GRK2 and GRK3 with much higher sensitivity to GRK3 than GRK2 [31–33]. The anti-GRK6 antibody labels both GRK5 and GRK6 proteins [31,33,34]. Due to the cross-reactivity of GRK3 and GRK6 antibodies we further confirmed the expression of the protein using western blot and mRNA by RT-PCR.
Regulation of the extrinsic and intrinsic apoptotic pathways in the prefrontal cortex of short- and long-term human opiate abusers
2008, NeuroscienceCitation Excerpt :These subjects were recruited as above (see healthy control subjects), including in all cases a negative screening for ethanol in blood samples. It is known that freezing, storage, and thawing can induce tissue sample damage (Grange-Midroit et al., 2002). In the prefrontal cortex of control subjects and opiate abusers, the uniform expression of the death substrate PARP-1 (116 kDa) (Fig. 9B) suggested the (antemortem) induction of little DNA damage, a good indicator of cell viability (Putt et al., 2005).