Keratinocyte-releasable factors increased the expression of MMP1 and MMP3 in co-cultured fibroblasts under both 2D and 3D culture conditions

G protein-coupled receptors (GPCRs) are known to modulate intracellular effectors involved in cardiac function. We recently reported homocysteine (Hcy)-induced ERK-phosphorylation was suppressed by pertussis toxin (PTX), which suggested the involvement of GPCRs in initiating signal transduction. An activated GPCR undergoes down regulation via a known mechanism involving ERK, GRK2, beta-arrestin1: ERK activity increases; GRK2 activity increases; beta-arrestin1 is degraded. We hypothesized that Hcy treatment leads to GPCR activation and down regulation. Microvascular endothelial cells were treated with Hcy. Expression of phospho-ERK1 and phospho-GRK2 was determined using Western blot, standardized to ERK1, GRK2, and beta-actin. Hcy was shown to dephosphorylate GRK2, thereby enhancing the activity. The results provided further evidence that Hcy acts as an agonist to activate GPCRs, followed by their down regulation. Hcy was also shown to decrease the content of the following G proteins and other proteins: beta-arrestin1, Galpha(q/11), Galpha(12/13), G(i/o).     

Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms

Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.     

Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor.

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.     

Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases

G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.     

Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization

Extracellular calcium rapidly controls PTH secretion through binding to the G protein-coupled calcium-sensing receptor (CASR) expressed in parathyroid glands. Very little is known about the regulatory proteins involved in desensitization of CASR. G protein receptor kinases (GRK) and beta-arrestins are important regulators of agonist-dependent desensitization of G protein-coupled receptors. In the present study, we investigated their role in mediating agonist-dependent desensitization of CASR. In heterologous cell culture models, we found that the transfection of GRK4 inhibits CASR signaling by enhancing receptor phosphorylation and beta-arrestin translocation to the CASR. In contrast, we found that overexpression of GRK2 desensitizes CASR by classical mechanisms as well as through phosphorylation-independent mechanisms involving disruption of Galphaq signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in beta-arrestin2 null mice, suggesting a functional role of beta-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and beta-arrestins play key roles in regulating CASR responsiveness in parathyroid glands.     

Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into beta-arrestin that were expected to disrupt two crucial elements that make beta-arrestin binding to receptors phosphorylation-dependent. We found that two beta-arrestin mutants (Arg169 --> Glu and Asp383 --> Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta2-adrenergic receptor (beta2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta-arrestin mutants effectively desensitize beta2AR in a phosphorylation-independent manner. Constitutively active beta-arrestin mutants also effectively desensitize delta opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type beta-arrestin + GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.     

MAPK-dependent degradation of G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling.     

G protein-coupled receptor kinases and beta arrestins are relocalized and attenuate cyclic 3',5'-adenosine monophosphate response to follicle-stimulating hormone in rat primary Sertoli cells.

The FSH receptor (FSH-R) is a member of the rhodopsin-like subfamily of G protein-coupled receptors that undergoes homologous desensitization upon agonist stimulation. In immortalized cell lines overexpressing the FSH-R, G protein-coupled receptor kinases (GRKs) and beta-arrestins are involved in the phosphorylation, uncoupling, and internalization of this receptor. In an effort to appreciate the physiological relevance of GRK/beta-arrestin actions in natural FSH-R-bearing cells, we used primary rat Sertoli cells as a model. GRK2, -3, -5, -6a, and -6b and beta-arrestins 1 and 2 were expressed in primary rat Sertoli cells. Overexpression of these different GRKs and beta-arrestins in primary rat Sertoli cells significantly attenuated the FSH-induced cAMP response, and FSH rapidly triggered a relocalization of endogenously expressed GRK2, -3, -5, and -6 and beta-arrestins 1 and 2 from the cytosol to the membranes. These results highlight the relationship existing between the GRK/beta-arrestin regulatory system and the FSH-R signaling machinery in a physiological model.     

RNA interference (RNA(i)) induction with various types of synthetic oligonucleotide duplexes in cultured human cells

G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use the extracellular signal-regulated kinase (ERK) cascade to regulate GRK2 cellular levels. ERK activation by receptor stimulation elevated endogenous GRK2 while antagonist treatment decreased cellular GRK2. Activating ERK by overexpressing constitutive active MEK-1 or Ras elevated GRK2 protein levels while blocking ERK using PD98059 or dominant negative Ras abolished this effect. These data suggest ERK is a critical regulator of GRK2 levels.     

Beta-arrestin2, a novel member of the arrestin/beta-arrestin gene family.

Homologous or agonist-specific desensitization of beta 2-adrenergic receptors (beta 2AR) is mediated by the beta-adrenergic receptor kinase (beta ARK) which specifically phosphorylates the agonist-occupied form of the receptor. However, the capacity of beta ARK-phosphorylated beta 2AR to stimulate Gs in a reconstituted system is only minimally impaired. Recently, a protein termed beta-arrestin, was cloned from a bovine brain cDNA library and found to quench phosphorylated beta 2AR-coupling to Gs. Utilizing a low stringency hybridization technique to screen a rat brain cDNA library, we have now isolated cDNA clones representing two distinct beta-arrestin-like genes. One of the cDNAs is the rat homolog of bovine beta-arrestin (beta-arrestin1). In addition, we have isolated a cDNA clone encoding a novel, beta-arrestin-related protein which we have termed beta-arrestin2. Overall, beta-arrestin2 exhibits 78% amino acid identity with beta-arrestin1. The primary structure of these proteins delineates a family of proteins that regulates receptor coupling to G proteins. The capacity of purified beta-arrestin1, beta-arrestin2, and arrestin to inhibit the coupling of phosphorylated receptors to their respective G proteins were assessed in a reconstituted beta 2AR-Gs system and in a reconstituted rhodopsin-GT system. beta-Arrestin2 was equipotent to beta-arrestin1 and specifically inhibited beta 2AR function. Conversely, arrestin inhibited rhodopsin coupling to GT, whereas beta-arrestin1 and beta-arrestin2 were at least 20-fold less potent in this system. beta-Arrestin1 and beta-arrestin2 are predominantly localized in neuronal tissues and in the spleen. However, low mRNA levels can be detected in most peripheral tissues. In the central nervous system, beta-arrestin2 appears to be even more abundant than beta-arrestin1. Immunohistochemical analysis of the tissue distribution of beta-arrestin1 and beta-arrestin2 in rat brain shows extensive, but heterogenous, neuronal labeling of the two proteins. They are found in several neuronal pathways suggesting that they have relatively broad receptor specificity regulating many G protein-coupled receptors. Furthermore, immunoelectron microscopy shows that the beta-arrestins are appropriately situated at postsynaptic sites to act in concert with beta ARK to regulate G protein-coupled neurotransmitter receptors.     

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.     

Changes in the expression of G protein-coupled receptor kinases and beta-arrestin 2 in rat brain during opioid tolerance and supersensitivity

We previously demonstrated that chronic treatment of rats with the mu-opioid receptor agonist sufentanil induced pharmacological tolerance associated with mu-opioid receptor desensitization and down-regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented mu-opioid receptor down-regulation, induced down-stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein-coupled receptor kinases (GRKs) and beta-arrestin 2 in agonist-induced mu-opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7-day chronic infusion of sufentanil (2 microgram/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 microgram/h) and nimodipine (1 microgram/h) for 7 days. Antinociception was evaluated by the tail-flick test. GRK2, GRK3, GRK6 and beta-arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up-regulation of GRK2, GRK6, and beta-arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and beta-arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and beta-arrestin 2 are involved in the short- and long-term adaptive changes in mu-opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and beta-arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.     

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.     

Cellular trafficking of G protein-coupled receptor/beta-arrestin endocytic complexes

beta-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta-arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta-arrestin2 in response to activation of several distinct GPCRs including the beta2-adrenergic receptor (beta2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta2AR activation, beta-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta-arrestins are confined to the periphery of cells and do not traffic along with activated beta2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta-arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta2AR carboxyl-terminal tail results in a beta-arrestin membrane localization pattern similar to that observed in response to beta2AR activation. In contrast, the corresponding chimeric beta2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/betaarrestin complexes and cellular distribution of beta-arrestins.     

G protein-coupled receptor kinase 2 modifies the cellular reaction to cisplatin through interactions with NADPH oxidase 4

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells—a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.

     

TGFβ-induced GRK2 expression attenuates AngII-regulated vascular smooth muscle cell proliferation and migration

Through diametric actions, the transforming growth factor β (TGFβ) and Angiotensin II (AngII) play important roles in regulating various biological responses such as cell proliferation and migration. Signaling initiated by TGFβ and AngII occurs through two structurally and functionally distinct receptor super families, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Previously, we identified the G protein-coupled receptor kinase-2 (GRK2), a key regulatory factor in the desensitization of GPCRs, as a direct downstream target of the TGFβ signaling cascade. GRK2 acts through a negative feed-back loop mechanism to terminate TGFβ-induced smad signaling. To investigate the impact of TGFβ-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). In this study, we show that activation of the TGFβ signaling cascade in VSMCs results in increased GRK2 expression levels, which consequently inhibits AngII-induced ERK phosphorylation and antagonizes AngII-induced VSMC proliferation and migration. Moreover, the inhibitory effect of TGFβ on AngII signaling occurs at the Mek-Erk interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFβ signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation and that GRK2 is a key factor mediating the cross-talk between these two receptor super families.     

Transient hypoxia differentially decreases GRK2 protein levels in CHO cells stably expressing the m1 mAChR

G protein-coupled kinase 2 (GRK2) has a key role in regulating signaling activities of a variety of G protein-coupled receptors (GPCRs). Several recent studies have directly implicated GRK2 phosphorylation in desensitization of GPCRs. In addition, binding by G(betagamma) or phosphorylation by PKC or c-Src [corrected] has been shown to activate or enhance GRK2 activity, respectively. Conversely, the calcium binding protein calmodulin or the serine/threonine kinase ERK has been implicated in inhibiting GRK2 activity. However, with the exception of a recent report indicating that activation of beta2-adrenergic receptor results in the ubiquitination and rapid degradation of GRK2, very little is known about cellular mechanisms that alter the protein levels of GRK2 [corrected]. Here, we report a novel serendipitous observation regarding alteration of GRK2 [corrected] protein levels. Exposure of CHO cells stably expressing the m1 muscarinic acetylcholine receptor (mAChR) to transient hypoxia caused near ablation of the GRK2 protein. In contrast, GRK2 protein levels remained unchanged in the parental CHO cells or in CHO cells stably expressing the m2 mAChR when exposed to transient hypoxia. The present study reports a novel observation that is unveiled by transient hypoxia in which GRK2 protein levels are altered by cellular mechanisms involving the m1 mAChR.     

Distinct Cellular and Subcellular Distributions of G Protein-Coupled Receptor Kinase and Arrestin Isoforms in the Striatum

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.