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Effect of Hypothyroidism on G Protein-Coupled Receptor Kinase 2 Expression Levels in Rat Liver, Lung, and Heart¹

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa (P.P., M.B., F.M.), and Instituto de Investigaciones Biomédicas, Alberto Sols, Consejo Superior de Investigaciones Científicas (M.A.-D., A.M.), Universidad Autónoma, 28049 Madrid, Spain

Address all correspondence and requests for reprints to: Federico Mayor, Jr., Centro de Biología Molecular, Universidad Autónoma, 28049 Madrid, Spain. E-mail: fmayor{at}cbm.uam.es

	Abstract

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GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of -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 (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 -adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or -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.

	Introduction

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THE ADRENERGIC system and thyroid hormones interact physiologically in a coordinated manner. In a variety of tissues, thyroid hormones enhance the -adrenergic receptor (AR)-mediated actions of catecholamines by increasing the accumulation of cAMP by mechanisms acting at both the receptor and postreceptor levels as well as by enhancing the transcriptional effects of cAMP. Hypothyroidism severely reduces the -adrenergic response in most tissues, contributing to the development of cardiovascular dysfunction, altered metabolic responses, and decreased thermogenesis (1, 2, 3, 4). Thyroid hormone deficit appears to promote such impaired responsiveness or sensitivity to catecholamines by reducing the number of AR and increasing the number of -adrenergic receptors. However, additional mechanisms are needed to explain the overall effect of hypothyroidism on adrenergic signal transduction (4).

Receptors for cathecolamines belong to the G protein-coupled receptor (GPCR) family. G protein-coupled receptor kinases (GRKs) are a family of serine-threonine kinases that specifically phosphorylate the agonist-occupied form of GPCR. This is followed by binding to the phosphorylated receptor of members of a second family of proteins, termed -arrestins, leading to receptor uncoupling from heterotrimeric G proteins and signal shut off. This process is known as desensitization, a general feature of GPCR signaling that involves a loss of receptor responsiveness after acute or sustained activation. GRK2 is one of the more abundant and broadly expressed GRKs that has been shown to participate in the regulation of adrenergic receptors and many other GPCRs (5, 6, 7). Interestingly, changes in the expression levels of GRK2 have been suggested to underlie alterations in adrenergic receptor signaling and desensitization in several physiopathological situations, such as heart failure, hypertension, experimental models of cardiac hypertrophy, or neonatal stress (7, 8, 9, 10, 11, 12, 13). In this context, we have explored the consequences of experimentally induced hypothyroidism on the protein expression levels of GRK2 at two developmental stages in several rat tissues (liver, heart, and lung) markedly affected by thyroid hormone deficit. We have also explored the expression levels of -adrenergic receptors; of GRK5, a member of the GRK family that modulates a variety of GPCR in the cardiovascular system (5, 6); and of -arrestin-1, a ubiquitous member of the -arrestin regulatory family. Our data indicate that hypothyroidism promotes significant and specific alterations in GRK2 levels in these tissues that may contribute to the previously reported changes in adrenergic receptor signaling in such pathological situations.

	Materials and Methods

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Animal treatment
Wistar rats raised in our animal facilities were used, and the rules of the European Community Council (directive of November 24, 1986, 86/609/ECC) concerning the maintenance and handling of animals were followed. Hypothyroidism was induced by a combination of chemical and surgical thyroidectomies, as previously described (14). To induce neonatal hypothyroidism, dams were given 0.02% methyl-mercaptoimidazol (Sigma, St. Louis, MO) in their drinking water from the ninth day after conception. Hypothyroidism in adult rats was induced by thyroidectomy at postnatal day 40 followed by 20 days of methyl-mercaptoimidazol treatment. Animals were killed at different postnatal ages by decapitation. Various tissues (lungs, hearts, and livers) were removed and frozen at -70 C until used.

Determination of GRK2, GRK5, and -arrestin-1 protein levels
Rat tissues were homogenized using a Polytron device (Brinkmann Instruments, Inc., Westbury, NY) in 4 vol 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM EGTA, and protein inhibitors (buffer A). The homogenate was centrifuged (1500 x g, 5 min, 4 C) to obtain a crude postnuclear supernatant. Aliquots of these lysates containing 100 µg protein were resolved in 7.5% or 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes for 60–75 min in 10 mM NaHCO₃, 3 mM Na₂CO₃ (pH 10), and 20% methanol using a Transblot cell (Bio-Rad Laboratories, Inc., Richmond, CA). The filters were blocked with 10 mM Tris-HCl (pH 7.5)/150 mM NaCl and 5% fat-free dried milk. GRK2 protein was detected with AbFP1, a polyclonal antibody raised against a fusion protein containing amino acids 50–145 of bovine GRK2, the specificity of which has been previously validated (15, 16). GRK5 protein levels were determined using both a commercially available affinity-purified antibody [GRK5 (C-20), Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution, 1:100] as well as a polyclonal antibody (Ab9) raised against recombinant GRK2 protein (13) that displays cross-reactivity with GRK5 (dilution, 1:1000). -Arrestin-1 expression was analyzed with Ab186, a polyclonal antibody raised in our laboratory against a fusion protein containing amino acids 172–268 of bovine -arrestin-1 that specifically recognizes this arrestin isoform (17, 18). Blots were developed using a chemiluminescent method (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) after incubation with a goat antirabbit antibody conjugated to peroxidase.

Radioligand binding assay of -adrenergic receptors
Rat tissues were homogenized with a Polytron device (three times, 30 sec each time) in 10 vol buffer A. The supernatant of a low speed centrifugation (2,000 x g, 4 min, 4 C) was centrifuged at 45,000 x g for 20 min at 4 C to obtain a plasma membrane pellet. Membranes were washed three times in buffer A and finally resuspended in the same buffer at concentrations of 3–10 mg protein/ml. Total AR number was determined as previously reported (13), using 5 nM [³H]dihydroalprenolol (Amersham Pharmacia Biotech) and 100 µM (-)propanolol to define nonspecific binding. ₁AR and ₂AR levels were estimated using 1 mM alprenolol as a specific ₁AR competitor. At least three different tissue samples from pools of three or four control or hypothyroid rats were employed for each binding determination; determinations were performed in triplicate.

	Results and Discussion

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To test the effect of hypothyroidism on GRK2 expression levels, we performed immunoblot analysis using extracts from selected rat tissues of normal and hypothyroid animals obtained at two different postnatal ages and a specific kinase antibody. In the heart, the normal developmental pattern of GRK2 expression is characterized by high levels at birth, followed by a sustained diminution until adulthood (Penela, P., unpublished data). Comparison of GRK2 immunoreactivity in the hearts of control and hypothyroid animals indicates that thyroid hormone deficiency significantly increases (1.5-fold compared with euthyroid animals) the expression of GRK2 at 60 days after birth, whereas the GRK2 expression levels attained on postnatal day 5 were similar in control and hypothyroid rats (Fig. 1). A significant increase in GRK5 protein levels was also observed in the heart of hypothyroid animals at 60 days after birth (Fig. 2), whereas the expression of the uncoupling protein -arrestin-1 was not altered at 5 or 60 days of postnatal development in the treated animals (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Figure 1. Protein levels of GRK2 in heart, lung, and liver of euthyroid and hypothyroid rats at different stages of postnatal development. Extracts from the indicated rat tissues (three independent pools of five or six animals) were obtained at two different postnatal ages from euthyroid and hypothyroid rats, as indicated in Materials and Methods. Proteins (100 µg/lane) were resolved by electrophoresis on 10% SDS-polyacrylamide gels, blotted to a nitrocellulose membrane, and probed with a specific antibody raised against GRK2 (AbFP1; dilution, 1:1000) as indicated in Materials and Methods, followed by densitometric analysis of the specific GRK2 bands. Expression levels in control conditions at each postnatal age are taken as 100%. Data are the mean ± SEM of six determinations. **, P < 0.01; ***, P < 0.0001 (compared with levels under euthyroid conditions at the corresponding age). , Control condition; , hypothyroid condition. Representative immunoblots are shown below.

View larger version (16K): [in this window] [in a new window]   Figure 2. Protein levels of GRK5 in adult heart and lung of euthyroid and hypothyroid rats. Tissue samples were obtained as described in Fig. 1, and proteins (75 µg/lane) were resolved by electrophoresis on 7.5% SDS-polyacrylamide gels. GRK5 expression was investigated by immunoblot as indicated in Materials and Methods, followed by densitometric analysis. The expression level in control conditions was taken as 100%. Data are the mean ± SEM of four to seven determinations. **, P < 0.01 compared with control levels. Representative immunoblots are shown. , Control condition; , hypothyroid condition.

View larger version (23K): [in this window] [in a new window]   Figure 3. Effects of hypothyroidism on -arrestin-1 protein levels in rat heart, lung, and liver at different stages of postnatal development. Samples were obtained as described in Fig. 1, and proteins (100 µg/lane) were resolved by electrophoresis on 10% SDS-polyacrylamide gels. -Arrestin-1 levels were analyzed with a specific polyclonal antibody (Ab186; dilution, 1:600) as detailed in Materials and Methods. Expression levels in control conditions at each postnatal age are taken as 100%. Data are the mean ± SEM of at least six determinations. **, P < 0.01 compared with levels under control conditions at the corresponding age. , Control condition; , hypothyroid condition. Representative immunoblots are shown below.

Increased GRK2 levels, as detected in the hearts of hypothyroid rats, would be predicted to impair -adrenergic receptor signaling. Interestingly, we found that both ₁AR and ₂AR levels are decreased at 60 days after birth in the hypothyroid rat heart (1AR, 32.2 ± 2.3 fmol/mg protein in control animals and 15.8 ± 6.5 fmol/mg protein in treated animals; ₂AR, 12.1 ± 6.4 fmol/mg protein in control rats and 7.6 ± 3.6 fmol/mg protein in hypothyroid animals; mean ± SEM of three to five independent experiments). It has been previously reported that ARs are markedly down-regulated in hypothyroid conditions (21, 22, 23), and that the sensitivity of cardiac cells to -agonist stimulation is decreased, particularly at high concentrations of agonist (21). Thus, hypothyroidism appears to simultaneously promote a reduction in AR density and an increase in GRK2 levels, leading to marked alterations in the receptor/GRK2 ratio and therefore to increased desensitization and impaired signaling of the remaining receptors. This situation is similar to that taking place in heart failure patients, where ₁AR reduction and increased GRK2 levels promote marked receptor desensitization and increased sympathetic tone (9, 24). In fact, similar alterations in catecholamine signaling and an increase in cardiac efferent sympathetic activity have also been reported as a consequence of T₃ deficiency (3). The fact that GRK5, a member of the GRK family involved in the modulation of GPCR present in cardiovascular cells (5, 6), also displays increased levels of expression in the hypothyroid rat heart would further lead to a decreased signaling mediated by -adrenergic and other GPCR in these circumstances. In summary, our data strongly suggest that the increased levels of GRKs in hypothyroid heart would contribute to the impairment of adrenergic responsiveness and cardiac function reported in such pathological situations.

Similar changes in GRK2 levels are also detected in the lung of hypothyroid rats at 60 days alter birth (Fig. 1). At this postnatal stage we do not detect significant changes in AR levels in the hypothyroid rat lung (data not shown), although a previous study reported diminished AR expression in the hypothyroid rats at 28 days of age (25). Overall, these data indicate that in the adult rat lung, T₃ deficiency modifies the normal pulmonary balance between ARs and their regulatory protein GRK2. Given that the expression level of this kinase has been correlated to the extent of -agonist-promoted desensitization in different pulmonary cell types (26), our findings suggest that the observed changes in GRK2 levels may indeed result in alterations of receptor function that may contribute to the impairments of surfactant release and smooth muscle relaxation commonly associated with thyroid disorders (4).

A different effect of thyroid hormone deficit on GRK2 levels is observed in the liver. In this tissue, GRK2 levels at birth are markedly high compared with adult values in control animals (13) (Penela, P., unpublished observations), and no GRK5 immunoreactivity is detected (not shown). Hypothyroidism appears to promote a further decrease in GRK2 immunoreactivity at 60 days of life (Fig. 1). At this stage, a significant, approximately 2-fold reduction in GRK2 expression is observed in hypothyroid rats. Such a decrease in GRK2 levels would be predicted to relieve GPCR desensitization, thus increasing responsiveness to agonists. Consistently, hypothyroidism has been shown to potentiate, rather than reduce, catecholamine action in this tissue compared with that in heart or lung. This effect was ascribed to a specific increase in AR number that would result in a preferential -agonist stimulation of gluconeogenesis and glycogenolysis (27, 28, 29), although there are controversial data on the effect of hypothyroidism in rat liver ARs depending on the rat strain and the methods of induction of hypothyroidism and radioligand binding employed (27, 30). In our hands, ₁AR levels were decreased (3-fold) and ₂AR number was increased (3.5-fold) in the hypothyroid rat liver (data not shown), consistent with the important role of ₂AR in mediating the actions of adrenaline in the liver under certain physiological conditions (31, 32). Hypothyroidism also promotes an improvement in AR coupling that cannot be ascribed to changes in adenylyl cyclase or G_s protein (7, 33, 34) and that could be explained by our reported changes in GRK2 expression. Interestingly, signaling through other GPCRs that play an essential role in hepatic metabolism, such as receptors for vasopressin or angiotensin II, is also altered in hypothyroid animals (35). Agonist stimulation of these receptors leads to a second messenger response similar to that of euthyroid controls despite the reduced number of receptors in such a condition, thus suggesting a better coupling of receptors to the transduction machinery, which would be favored by reduced GRK2 levels.

The observed changes in -arrestin-1 expression in lung and liver are opposed to those found for GRK2 levels in these tissues, confirming an independent regulation of the expression of these proteins and suggesting the occurrence of compensatory mechanisms. However, the potential functional correlates of these changes in -arrestin-1 levels in the hypothyroid animals are more difficult to discuss, given the fact that this protein acts downstream of GRK2 in GPCR regulation and that each -arrestin isoform can display different affinities for activated GPCR (for instance, -arrestin-1 binding to ₁AR is weaker than that to ₂AR) (36). More detailed studies will be needed to ascertain how the combined changes in GRK and -arrestin expression in a given tissue or cell type affect the function of specific GPCRs in hypothyroidism.

In summary, we found that experimental hypothyroidism leads to selective changes in GRK2 expression in several rat tissues, thus suggesting that thyroid hormones are able to directly or indirectly regulate GRK2 protein levels in a tissue-specific manner by mechanisms that need to be addressed in future studies. The observed changes in GRK2 levels in hypothyroid animals are strikingly correlated with previously reported changes in adrenergic signaling in this physiopathological condition. Our results suggest that even modest expression changes at the levels of both GPCR and their regulatory protein GRK2 would lead to significant alterations of the receptor/GRK2 ratio in hypothyroidism and may contribute to a better understanding of the impairment of -adrenergic and GPCR signaling that underlies thyroid disorders.

	Acknowledgments

 
The authors thank A. Morales for skillful secretarial assistance.

	Footnotes

 
¹ This work was supported by grants (to F.M.) from the Ministerio de Educación y Cultura (PM98-0020), the Comunidad de Madrid (8.4.6.98), and the European Union (BMH4-3596) and a grant (to A.M.) from Plan Nacional (SAF98-0060). The Centro de Biología Molecular Severo Ochoa holds and institutional grant from Fundación Ramón Areces.

² Recipients of a postdoctoral fellowship from Comunidad de Madrid.

Received October 10, 2000.

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