Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and ß-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. ß-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes.

Diagnostic value of interleukin-12 p40 in tuberculous pleural effusions.

The diagnosis of tuberculous pleural effusion (TBPE) is frequently problematic. Several markers of TBPE in pleural fluid have been evaluated, with different results. Pleural effusions from 96 patients were classified on the basis of definitive diagnosis as tuberculous (n = 39), neoplastic (n = 42) or parapneumonic (n = 15). Adenosine deaminase (ADA), ADA isoform ADA-2, interferon (IFN)-gamma, CD3(+)/DR(+) T-lymphocytes and interleukin (IL)-12 p40 were determined in all 96 effusions. The efficiency of IL-12 p40 for diagnosis of TBPEs was evaluated, in comparison with those of the other parameters, by comparing the areas under their receiver operating characteristics. With the threshold value of 550 pg.mL(-1), IL-12 p40 had a sensitivity of 92.3% (36 out of 39) and specificity of 70.2% (17 false positives). The misclassification rate of IL-12 p40 was significantly greater than those of ADA-2 and ADA. Among TBPEs, ADA correlated significantly with ADA-2, and IFN-gamma with ADA and IL-12 p40. Although tuberculous pleural effusions show values of interleukin-12 p40 that are significantly higher than neoplastic and parapneumonic fluids, this parameter is less efficient than adenosine deaminase, adenosine deaminase isoform 2 and interferon-gamma. Its routine determination is, accordingly, not justified.

Hydrogen peroxide impairs GRK2 translation via a calpain-dependent and cdk1-mediated pathway.

Oxidative mechanisms of injury are involved in many neurodegenerative diseases such as stroke, ischemia-reperfusion injury and multiple sclerosis. G protein-coupled receptor kinase 2 (GRK2) plays a key role in G protein-coupled receptor (GPCR) signaling modulation, and its expression levels are decreased after brain hypoxia/ischemia and reperfusion as well as in several inflammatory conditions. We report here that hydrogen peroxide downregulates GRK2 expression in C6 rat glioma cells. The hydrogen peroxide-induced decrease in GRK2 is prevented by a calpain protease inhibitor, but does not involve increased GRK2 degradation or changes in GRK2 mRNA level. Instead we show that hydrogen peroxide treatment impairs GRK2 translation in a process that requires Cdk1 activation and involves the mTOR pathway. This novel mechanism for the control of GRK2 expression in glial cells upon oxidative stress challenge may contribute to the modulation of GPCR signaling in different pathological conditions.

Beta-arrestin- and c-Src-dependent degradation of G-protein-coupled receptor kinase 2.

G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. We recently have described that GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). In this report, we find that beta-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of beta-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with beta-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling.

Effect of hypothyroidism on G protein-coupled receptor kinase 2 expression levels in rat liver, lung, and heart.

GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of beta-adrenergic and other G protein-coupled receptors and plays an important role in their desensitization and modulation. Alterations in thyroid hormone levels have been reported to lead to important changes in adrenergic receptor responsiveness and signaling in a variety of tissues. In this context, we have explored the effects of experimental hypothyroidism on GRK2 protein levels in rat heart, lung, and liver using a specific antibody. Hypothyroid animals show significant up-regulation ( approximately 50% increase compared with controls) in GRK2 levels in heart and lung at 60 days after birth, whereas a 50% reduction is detected in the liver at this stage. These alterations are selective, as beta-adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or beta-arrestin-1, display a different pattern of expression changes in the hypothyroid animals. The reported changes in GRK2 levels and in the receptor/kinase ratio predict alterations in adrenergic receptor desensitization and signal transduction efficacy consistent with those observed in thyroid disorders, thus suggesting a relevant role for the modulation of GRK2 expression in this physiopathological condition.

Expression of the G protein-coupled receptor kinase 2 during early mouse embryogenesis.

Whilst several G protein-coupled receptors (GPCRs) have been shown to play important roles during development, little study has been carried out on the G protein-coupled receptor kinases (GRKs) that modulate their activity in embryos. Here, we have analyzed the expression of GRK2, the predominant GRK expressed during embryogenesis. We show that at early stages, the expression of GRK2 is restricted to populations of cells that are undifferentiated, multipotent and in many cases, migratory. As such, GRK2 transcripts were found in the early mesoderm and neural crest as they migrate from the primitive streak and the neural tube, respectively. In the limb bud, GRK2 transcripts were observed in cells of the progress zone and in the interdigital areas. At later stages, the expression in the heart is compatible with the phenotype observed in the GRK2 deficient mice.

Expression patterns of the regulatory proteins G protein-coupled receptor kinase 2 and beta-arrestin 1 during rat postnatal brain development: effect of hypothyroidism.

G protein-coupled receptor kinase 2 (GRK2) and beta-arrestin 1 are key regulatory proteins that modulate the desensitization and resensitization of a wide variety of G protein-coupled receptors (GPCRs) involved in brain functions. In this report, we describe the postnatal developmental profile of the mRNA and protein levels of GRK2 and beta-arrestin 1 in rat brain. The expression levels of GRK2 and beta-arrestin 1 display a marked increase at the second and third week after birth, respectively, consistent with an involvement of these proteins in brain maturation processes. However, the expression attained at birth and during the first postnatal week with respect to adult values (45-70% for GRK2, approximately 30% for beta-arrestin 1) is relatively high compared to that reported for several GPCRs, indicating the existence of changes in the ratio of receptors to their regulatory proteins during brain development. On the other hand, we report that experimental hypothyroidism results in changes in the patterns of expression of GRK2 and beta-arrestin 1 in cerebral cortex, leading to a 25-30% reduction in GRK2 levels at several stages of development. Such changes could help to explain the alterations in GPCR signaling that occur during this pathophysiological condition.

Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCR) is emerging as an important feature of several cardiovascular diseases. G protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of a variety of these receptors, and its cardiac expression levels are altered in pathological situations such as chronic heart failure. However, very little is known about the signals and mechanisms that modulate GRK2 expression in cardiovascular cells. METHODS AND RESULTS: We have studied the transcriptional activity of the 1.6-kb-long proximal genomic region of the human GRK2 gene. In an aortic smooth muscle cell line, agents that lead to physiological vasoconstriction and hypertrophy, such as phorbol esters, increased GRK2 promoter activity. Activation of signaling pathways by cotransfected G(alphaq) subunits or alpha(1)-adrenergic receptors also markedly enhanced the expression of the GRK2 promoter constructs. Conversely, proinflammatory cytokines, such as interleukin-1beta, tumor necrosis factor-alpha, or interferon-gamma, led to the opposite effect, decreasing the activity of the GRK2 promoter. CONCLUSIONS: Our results suggest that the expression of GRK2 in vascular cells is tightly controlled at the transcriptional level by the interplay between several extracellular messengers, which may trigger alterations of normal GRK2 levels in some physiopathological circumstances, thus promoting changes in the efficacy of the GPCR signal transduction.

Lymphocyte populations in tuberculous pleural effusions.

Different systemic and local responses to mycobacterial antigens suggest an active compartmentalization of responsive lymphocytes to tubercular antigens. This fact, observed in pleuritic processes, raises doubts about the accuracy of information obtained in the study of cells taken solely from peripheral blood. For this reason we decided to study the concept of compartmentalization in 140 patients suffering from pleural effusions. Patients were classified into six groups according to the aetiology of the effusion: group I, tuberculous, n = 23; group II, paraneoplastic, n = 41; group III, metapneumonic empyematous, n = 5; group IV, transudate, n = 38; group V, miscellaneous exudate, n = 19; group VI, unknown aetiology, n = 14. In each group we studied the lymphocyte population by using flow cytometry with doubly fluorescent monoclonal antibodies: B [expressing human lymphocyte antigen (HLA)-DR on the surface], T (CD3+), CD4+ and CD8+, and the subpopulation of activated T lymphocytes (together expressing CD3 and HLA-DR on the surface) (CD3+DR+). The study of these subpopulations in peripheral blood did not yield valuable results, but the CD3+DR+ population in pleural fluid demonstrated a diagnostic efficiency of 84% [positive predictive value (PPV) 51%, negative predictive value (NPV) 96%] at a cut-off value of 80.4 cells/mm3. The CD3+DR+ pleural fluid/peripheral blood ratio demonstrated an efficiency of 83% (PPV 50%, NPV 96%), and showed a statistically significant difference (P < 0.02) with regard to all the diagnostic groups, with the exception of the paraneoplastic effusions. The lymphocytic subpopulations study confirms the concept of compartmentalization in tuberculous pleuritis, as shown by the greater number of activated T lymphocytes present in pleural fluid in comparison with peripheral blood in tuberculous pleuritis, a 98% efficiency of adenosine deaminase (ADA) determination in pleural fluid versus a 50% value in peripheral blood, predominance of helper cells (CD4+) in pleural fluid and suppressor cells (CD8+) in peripheral blood, a greater CD4+/CD8+ ratio in pleural fluid than in peripheral blood, and a significant correlation of ADA-CD3+DR+ in pleural fluid, which does not occur in peripheral blood.

Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway.

GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family and has been shown to play a key role in determining the desensitization and resensitization patterns of a variety of G protein-coupled receptors. In this report, we show that GRK2 is actively degraded by the proteasome proteolytic pathway, unveiling a new mechanism for the rapid regulation of its expression levels. Interestingly, activation of beta2-adrenergic receptors (beta2AR) markedly increases GRK2 ubiquitination and degradation through the proteasome pathway. In addition, blocking GRK2 degradation notably alters beta2AR signaling and internalization, consistent with a relevant physiological role for GRK2 proteasomal degradation. Activity-dependent modulation of GRK2 cellular levels emerges as an important mechanism for modulating the cellular response to agonists acting through G protein-coupled receptors.

Tuberculous pleurisy: a study of 254 patients.

OBJECTIVES: To determine the age at which tuberculous pleural effusions occur, the radiological and biochemical characteristics of the effusions, the sensitivities of the various diagnostic tests, and the utility of combining clinical, radiological, and analytic data in diagnosis. METHODS: We studied the case histories of 254 patients in whom tuberculous pleural effusions were diagnosed with certainty between January 1, 1989, and June 30, 1997, in a Spanish university hospital in a region with a high incidence of tuberculosis. RESULTS: The mean (+/-SD) age of the patients was 34.1+/-18.1 years, and 62.2% were younger than 35 years. The effusion was on the right side in 55.9% of patients, on the left side in 42.5% of patients, and on both sides in 1.6% of patients. In 81.5% of patients, less than two thirds of the hemithorax was affected. Associated pulmonary lesions were detected in 18.9% of patients, of whom 14.6% exhibited cavitation. In 93.3% of the effusions, more than 50% of leukocytes were lymphocytes, and almost all had the biologic characteristics of exudates (98.8% had high total protein contents, 94.9% had high cholesterol levels, and 82.3% had high lactate dehydrogenase levels). All but 1 effusion (99.6%) had an adenosine deaminase (ADA) concentration higher than 47 U/L, 96.8% (123/127) of the effusions had high ADA2 levels, and 89% (73/82) of the effusions had high interferon gamma levels. Adenosine deaminase 2 contributed 72.2%+/-12.5% (mean +/- SD) of total ADA activity. Total ADA activity was significantly correlated with ADA2 (r = 0.83) and with interferon gamma (r = 0.30) levels. Definitive diagnosis was based on the observation of caseous granulomas in pleural biopsy tissue samples in 79.8% of patients, on the results of biopsy cultures in 11.7% of patients, and on pleural effusion cultures in the remaining 8.5% of patients. Results of the tuberculin skin test were positive in only 66.5% of patients. CONCLUSIONS: In these patients, lymphocyte-rich exudative pleural effusions occurred, on average, at a young age, with no preference for either the right or the left side; normally affected no more than two thirds of the hemithorax; and were generally unaccompanied by pulmonary infiltrates. High ADA concentration was a highly sensitive diagnostic sign and was caused by a rise in ADA2 concentration. The most sensitive criterion based on pleural biopsy was the observation of caseous granulomas, and culture of biopsy material further increased overall sensitivity. Negative skin test results were no guarantee of the effusion being nontuberculous. This, together with the low mean age of the patients and the low frequency of associated pulmonary lesions, suggests that tuberculous pleural effusion is a primary form of tuberculosis in this region.

The subcellular and cellular distribution of G protein-coupled receptor kinase 2 in rat brain.

G protein-coupled receptor kinase 2 has been found to phosphorylate and thus regulate the activity of several G protein-coupled receptors implicated in neuronal signalling pathways. Although this kinase was initially described as a soluble protein, our laboratory has recently found that a significant amount of G protein-coupled receptor kinase 2 is associated with microsomal membranes in liver and different types of cultured cells. In the present report we show that high G protein-coupled receptor kinase 2 specific activity and protein levels are present in microsomal fractions of rat brain homogenates. On the other hand, immunochemical detection using a new antibody raised against the N-terminus of the kinase revealed a specific and widely distributed staining in different areas of the central nervous system, and the association of G protein-coupled receptor kinase 2 with intracellular structures in nervous cells. Our results further suggest that this receptor kinase may be involved in the modulation of G protein-coupled receptor-mediated neurotransmission and that association with microsomal membranes may play a role in G protein-coupled receptor kinase 2 functions in the brain.

G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

Role of G protein-coupled receptor kinase 2 and arrestins in beta-adrenergic receptor internalization.

G protein-coupled receptors (GPCRs) mediate the action of messengers that are key modulators of the function, growth, and differentiation of cardiac and vascular cells. A general feature of GPCRs is the existence of complex regulatory mechanisms that modulate receptor responsiveness and underlie important physiologic phenomena such as signal integration and desensitization. The molecular mechanisms of desensitization have been investigated with the beta2-adrenergic receptor (beta2AR) used as the main model system. Rapid regulation of betaAR and other GPCRs appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization, which plays a key role in resensitizing GPCR by allowing its dephosphorylation and recycling. Recent data indicate that, besides the uncoupling function, GRK2 and beta-arrestin also directly participate in beta2AR sequestration, thus providing the trigger for its resensitization. A detailed knowledge of the role of GRKs and arrestins in betaAR internalization would make their physiologic role in the modulation of cellular responses to messengers better understood.

Utility of tumour markers in the diagnosis of neoplastic pleural effusion.

Approximately 20% of pleural effusions are caused by neoplastic processes. Although cytology is the most specific routine diagnostic procedure, its sensitivity of 50-60% is insufficient, and thus diagnosis is usually carried out by more invasive techniques such as pleural biopsy, thoracoscopy or thoracotomy. The object of this study is to evaluate the use of determining some tumour markers in pleural fluid obtained by thoracocentesis for diagnosis of neoplastic pleural effusion. Patients (271) with pleural effusions were classified in five groups: I: neoplasms n = 88; II: tuberculosis n = 63; III: parapneumonics n = 53; IV: miscellaneous exudates n = 39 and V: transudates n = 28. The tumour markers studied were: carcinoembryonic antigen (CEA), CA 125, squamous cell carcinoma antigen (SCC), and neuron specific enolase (NSE). The tumour makers had the following diagnostic efficiencies for neoplastic origin of the pleural effusion: CEA 76% (sensitivity 31%, specificity 93%); CA 125 66% (70% and 61%); SCC 65% (48% and 80%) and NSE 53% (30% and 89%). The diagnostic efficiencies for pulmonary neoplastic origins were 68% for NSE (sensitivity 83%, specificity 53%); 65% for SCC (54% and 75%); 63% for CEA (80% and 48%) and 61% for CA 125 (79% and 42%). We believe that the routine testing of tumour markers in pleural fluid obtained by thoracocentesis would greatly increase diagnostic effectiveness and could avoid the practice of more aggressive diagnostic techniques on the patient.

Association of the regulatory beta-adrenergic receptor kinase with rat liver microsomal membranes.

beta-Adrenergic receptor kinase (beta ARK) is a regulatory enzyme involved in the modulation of beta-adrenergic and other G protein-coupled receptors. It has been described that beta ARK is a cytosolic protein that transiently translocates to the plasma membrane in order to specifically phosphorylate agonist-occupied receptors. In this report, we used beta ARK-specific antibodies to demonstrate that a significant amount of this kinase is present in rat liver microsomal membranes. beta ARK seems to be peripherally associated with the cytosolic side of microsomal membranes since it can be stripped from the membranes by mild salt treatment. Cell-free association experiments indicate that the interaction of beta ARK is reversible, saturable, and strongly inhibited by protease or heat treatment of the microsomes, thus suggesting that beta ARK interacts with a protein component of the microsomal membrane. Gradient fractionation studies indicate that the highest beta ARK-specific activity co-migrates with endoplasmic reticulum enzymatic markers. Furthermore, indirect immunofluorescence and immunogold electron microscopy experiments performed in cultured cells using affinity-purified anti-beta ARK antibodies are consistent with this subcellular localization pattern. Taken together, our data suggest that several beta ARK pools (i.e. microsome-bound, plasma membrane-bound, and cytosolic) may exist inside the cell. Such results are in line with recent reports showing that proteins involved in plasma membrane signal transduction, such as heterotrimeric G proteins, are also associated with membranes of different intracellular organelles.