Research report
G protein-coupled receptor kinases, β-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution

https://doi.org/10.1016/S0169-328X(02)00144-4Get rights and content

Abstract

G protein-coupled receptor kinases (GRKs) and β-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK2, GRK6, β-arrestin-2 and associated regulatory proteins (Gβ proteins and protein phosphatase (PP)-2A) were quantitated in human brains (immunodensity with specific antibodies) to assess for postmortem changes (pattern of protein degradation) and to investigate the effect of aging on these regulatory proteins as well as their subcellular distribution (cytosol and membrane fractions). In brain (prefrontal cortex, total homogenate) of healthy subjects (n=14) the immunodensities of GRK2 (r=−0.76), GRK6 (r=−0.64), β-arrestin-2 (r=−0.57), Gβ proteins (r=−0.59) and neurofilament (NF)-L (r=−0.64), but not PP-2A, declined markedly with the length of postmortem delay (PMD, 3–81 h). With these linear decay models, the average decreases per 12 h of PMD (from 12 to 72 h) were 7–11% for the various proteins. The immunodensities of GRK2 (r=−0.71), GRK6 (r=−0.61), and β-arrestin-2 (r=−0.54) in human brain (n=12) also declined with aging (16 to 87 years) and the average decreases per decade (from 20 to 80 years) were 3–5%. In contrast, the immunodensities of PP-2A, Gβ and NF-L in brain did not correlate significantly with the age of the subject at death (16–87 years). The immunodensities of GRK2/6 and β-arrestin-2 showed marked individual variations and were strongly reduced after several freeze/thaw cycles. In the prefrontal cortex the subcellular distribution (cytosol/membrane) of the two GRKs differed markedly (GRK2: 60%/40%; GRK6: 5%/95%), and that of β-arrestin-2 was as expected for a soluble protein (60%/40%). In brains of healthy subjects, the immunodensities of cytosolic GRK2 and β-arrestin-2 correlated, respectively, with those of membrane-associated GRK2 (r=0.67, P=0.049, n=9) and membrane-associated β-arrestin-2 (r=0.77, P=0.01, n=9). The results of this study emphasize the importance of examining relevant variables (PMD, age) and potential artifacts (individual variation, freeze–thawing effect) when designing signal transduction studies in neuropsychiatric disorders using the postmortem human brain.

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).

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