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Biomedical subjects

M J Lohse

Publications and source records attributed to M J Lohse.

At least 19 recordsLinked to original sources
PubMed2004

Hypoglycemia-dependent beta2-adrenoceptor downregulation: a contributing factor to hypoglycemia unawareness in patients with Type-1 diabetes?

To evaluate the influence of the incidence and unawareness of hypoglycemia on lymphocyte beta2-adrenoceptor densities, we measured beta2-adrenoceptor density using [125I]-iodocyanopindolol and CGP 12177 before and after 1 week of treatment optimization in 33 adults with type-1 diabetes mellitus. Diabetes treatment of all patients was modified to improve their glycemic control. During this week, all patients had to complete a protocol with 7 daily glucose measurements, one of which was at night. The subjective symptoms were evaluated in case of hypoglycemia. A significant correlation between a hypoglycemia incidence below (but not above) the threshold of 2.75 mmol/l (50 mg/dl) and beta2-adrenoceptor densities on lymphocytes was found after the study week (r = -0.72, p < 0.00001). Nine patients suffering from hypoglycemia unawareness had a significantly higher incidence of hypoglycemia (p < 0.002) and lower beta2-adrenoceptor densities on lymphocytes compared to 24 patients who recognized all of their hypoglycemic episodes (p < 0.004). We conclude that downregulation of beta2-adrenoceptor densities on lymphocytes occurs as a result of recurrent hypoglycemia defined as glucose levels of < 2.75 mmol/l. Beta2-adrenoceptor densities are decreased in patients with subjective hypoglycemia unawareness and might contribute to the reduced beta-adrenergic sensitivity in this subgroup of patients.

Adolescent↗
PubMed2004

Comparative pharmacology of human beta-adrenergic receptor subtypes--characterization of stably transfected receptors in CHO cells.

Although many beta1-receptor antagonists and beta2-receptor agonists have been used in pharmacotherapy for many years their pharmacological properties at all three known subtypes of beta-adrenergic receptors are not always well characterized. The aim of this study was, therefore, to provide comparative binding characteristics of agonists (epinephrine, norepinephrine, isoproterenol, fenoterol, salbutamol, salmeterol, terbutalin, formoterol, broxaterol) and antagonists (propranolol, alprenolol, atenolol, metoprolol, bisoprolol, carvedilol, pindolol, BRL 37344, CGP 20712, SR 59230A, CGP 12177, ICI 118551) at all three subtypes of human beta-adrenergic receptors in an identical cellular background. We generated Chinese hamster ovary (CHO) cells stably expressing the three beta-adrenergic receptor subtypes at comparable levels. We characterized these receptor subtypes and analyzed the affinity of routinely used drugs as well as experimental compounds in competition binding studies, using the non-selective antagonist 125I-cyanopindolol as a radioligand. Furthermore, we analyzed the beta-receptor-mediated adenylyl cyclase activity in isolated membranes from these cell lines. The results from our experiments show that all compounds exhibit distinct patterns of selectivity and activity at the three beta-receptor subtypes. In particular, a number of beta2- or beta3-receptor agonists that are inverse agonists at the other subtypes were identified. In addition, beta1-receptor antagonists with agonistic activity at beta2- and beta3-receptors were found. These specific mixtures of agonism, antagonism, and inverse agonism at different subtypes may have important implications for the therapeutic use of the respective compounds.

Adenylyl Cyclases↗
PubMed2004

Kinetics of G-protein-coupled receptor signalling and desensitization.

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor-arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.

Animals↗
PubMed2003

Effects of two Gbetagamma-binding proteins--N-terminally truncated phosducin and beta-adrenergic receptor kinase C terminus (betaARKct)--in heart failure.

Myocardial overexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct) has been shown to result in a positive inotropic effect or an improvement of survival in heart failure. However, it is not clear whether this beneficial effect is mainly because of dominant-negative inhibition of betaARK1, and a consecutive resensitization of beta-adrenergic receptors (betaAR), or rather due to inhibition of other Gbetagamma-mediated effects. In this study, we tested whether overexpression of N-terminally truncated phosducin (nt-del-phosducin), another Gbetagamma-binding protein that does not resensitize betaARs owing to simultaneous inhibition of GDP release from Galpha subunits, shows the same effects as betaARKct. Adenoviral gene transfer was used to express nt-del-phosducin and betaARKct in isolated ventricular cardiomyocytes and in myocardium of rabbits, which suffered from heart failure because of rapid ventricular pacing. BetaAR-stimulated cAMP formation was increased by betaARKct, but not by nt-del-phosducin, whereas both proteins inhibited Gbetagamma-mediated effects. Both transgenes also increased contractility of normal and failing isolated cardiomyocytes and improved contractility in rabbits with heart failure after gene transfer in vivo. In conclusion, overexpression of nt-del-phosducin enhances the contractility of cardiomyocytes to the same extent as betaARKct, suggesting that the effects of betaARKct might be owing to inhibition of Gbetagamma rather than to betaAR resensitization.

Adenoviridae↗
PubMed2001

Vascular hypertrophy and increased P70S6 kinase in mice lacking the angiotensin II AT(2) receptor.

BACKGROUND: Angiotensin II activates 2 distinct G protein-coupled receptors, the AT(1) and AT(2) receptors. Most of the known cardiovascular effects of angiotensin II are mediated by the AT(1) receptor subtype. The aim of the present study was to test whether deletion of the AT(2) receptor gene in mice (AT(2)-KO mice) leads to long-term functional or structural alterations in the cardiovascular system. METHODS AND RESULTS: In vivo pressure responses to angiotensin II or the alpha(1)-adrenergic receptor agonist phenylephrine were greatly enhanced in AT(2)-KO mice. Deletion of the angiotensin AT(2) receptor did not lead to a compensatory increase of the activity of the circulating renin-angiotensin system, and arterial blood pressure was identical in wild-type control mice (WT) and AT(2)-KO mice. Cardiac contractility as assessed by LV catheterization and by rapid MRI also did not differ between AT(2)-KO and WT mice. Isolated femoral arteries from AT(2)-KO mice, however, showed enhanced vasoconstriction to angiotensin II, norepinephrine, and K(+) depolarization compared with WT. Morphometric analysis of large and small femoral arteries revealed a significant hypertrophy of media smooth muscle cells. Phospho-P70S6 kinase levels were significantly increased in aortas from AT(2)-KO mice compared with WT mice. Treatment of mice with an ACE inhibitor for 8 weeks abolished the increased pressure responsiveness, vascular hypertrophy, and enhanced P70S6 kinase phosphorylation in AT(2)-KO mice. CONCLUSIONS: These results indicate that vascular AT(2) receptors inhibit the activity and, hence, hypertrophic signaling by the P70S6 kinase in vivo and thus are important regulators of vascular structure and function.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

Early impairment of calcium handling and altered expression of junctin in hearts of mice overexpressing the beta1-adrenergic receptor.

Chronic stimulation of cardiac beta1-adrenergic receptors contributes to disease progression and mortality in patients and animal models of heart failure. To search for the mechanism of adrenergic impairment of cardiac function in vivo, we studied transgenic mice with cardiac-specific overexpression of beta1-adrenergic receptors. Transgenic mice with cardiac overexpression of beta1-adrenergic receptors showed progressive left ventricular fibrosis starting at 4 months of age. Left ventricular catheterization revealed a modest enhancement of contractility and relaxation at 2 months of age, followed by progressive dysfunction in both parameters and ultimately cardiac failure. When the effects of endogenous catecholamines were blocked by the b-receptor antagonist propranolol, maximal rate of contractility (dp/dtmax) and maximal rate of relaxation (dp/dtmin) were significantly blunted in 2-month-old beta1-receptor transgenic mice. Isolated cardiomyocytes from these animals displayed markedly altered calcium transients with significant prolongation of the intracellular calcium transient compared with nontransgenic littermates. We determined the expression of sarcoplasmic reticulum proteins involved in calcium handling by RNase protection assay and by immunoblotting. Although the expression of calsequestrin, triadin, and phospholamban was not altered, we observed a progressive decrease in junctin abundance in beta1-receptor transgenic mice (Pbeta1-adrenergic receptors.

Animals↗
PubMed2001

Activation and deactivation kinetics of alpha 2A- and alpha 2C-adrenergic receptor-activated G protein-activated inwardly rectifying K+ channel currents.

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from alpha(2A)-adrenergic receptor knockout mice suggested that the alpha(2A)-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the alpha(2C)-adrenergic receptor. Here we investigated whether these functional differences between presynaptic alpha(2)-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. alpha(2A)- and alpha(2C)-receptors were stably expressed in HEK293 cells at moderate ( approximately 2 pmol/mg) or high (17-24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K(+) (GIRK) channels was similar in extent and kinetics for alpha(2A)- and alpha(2C)-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. alpha(2C)-Receptor-activated GIRK currents returned much more slowly to base line than did alpha(2A)-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine alpha(2C)- than at the alpha(2A)-receptor subtype and may explain why alpha(2C)-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the alpha(2A)-receptor subtype.

Action Potentials↗
PubMed2001

Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein.

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.

Animals↗
PubMed2001

Dobutamine-stress magnetic resonance microimaging in mice : acute changes of cardiac geometry and function in normal and failing murine hearts.

The aim of this study was to assess the capability of MRI to characterize systolic and diastolic function in normal and chronically failing mouse hearts in vivo at rest and during inotropic stimulation. Applying an ECG-gated FLASH-cine sequence, MRI at 7 T was performed at rest and after administration of 1.5 microgram/g IP dobutamine. There was a significant increase of heart rate, cardiac output, and ejection fraction and significant decrease of end-diastolic and end-systolic left ventricular (LV) volumes (P<0.01 each) in normal mice during inotropic stimulation. In mice with heart failure due to chronic myocardial infarction (MI), MRI at rest revealed gross LV dilatation. There was a significant decrease of LV ejection fraction in infarcted mice (29%) versus sham mice (58%). Mice with MI showed a significantly reduced maximum LV ejection rate (P<0.001) and LV filling rate (P<0.01) and no increase of LV dynamics during dobutamine action, indicating loss of contractile and relaxation reserve. In 4-month-old transgenic mice with cardiospecific overexpression of the beta(1)-adrenergic receptor, which at this early stage do not show abnormalities of resting cardiac function, LV filling rate failed to increase after dobutamine stress (transgenic, 0.19+/-0.03 microL/ms; wild type, 0.36+/-0.01 microL/ms; P<0.01). Thus, MRI unmasked diastolic dysfunction during dobutamine stress. Dobutamine-stress MRI allows noninvasive assessment of systolic and diastolic components of heart failure. This study shows that MRI can demonstrate loss of inotropic and lusitropic response in mice with MI and can unmask diastolic dysfunction as an early sign of cardiac dysfunction in a transgenic mouse model of heart failure.

Adrenergic beta-Agonists↗
PubMed2001

Immunofluorescent imaging of beta 1- and beta 2-adrenergic receptors in rat kidney.

BACKGROUND: beta-Adrenergic receptors (beta-ARs) are known to participate in the regulation of glomerular filtration, NaCl reabsorption, acid-base balance, and renin secretion; however, the precise histologic localization of beta-AR at putative signaling sites involved in these processes remains an open issue. METHODS: We used a set of subtype-specific rabbit antibodies to visualize beta(1)- and beta(2)-AR in rat kidney by immunohistochemistry and specified cells and segments of the nephron thought to be regulated by catecholamines. In addition, the relative proportion of beta-AR subtypes in cortical and medullary portions of rat kidney was determined by Western blotting and by competing [(125)I]-cyanopindolol binding with the beta(1)- or beta(2)-selective antagonists bisoprolol or ICI 118,551, respectively. RESULTS: Immunoreactivity for beta(1)-AR was found in mesangial cells, juxtaglomerular granular cells, the macula densa epithelium, proximal and distal tubular segments, and acid-secreting type A intercalated cells of the cortical and medullary collecting ducts. Immunoreactivity for beta(2)-AR was predominantly localized in the apical and subapical compartment of proximal and, to a lesser extent, distal tubular epithelia (suggesting interactions with luminal fluid catecholamines). Both subtypes were dense in the membranes of smooth muscle cells from renal arteries. Concordant data were obtained by radioligand binding and immunoblotting of membranes prepared from cortical and medullary portions of the kidney. CONCLUSION: Our data provide an immunohistochemical basis for the cellular targets of beta-adrenergic regulation of renal function. Moreover, they could help to devise therapeutic strategies directed at renal beta-ARs.

Animals↗
PubMed2001

Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta(1)- or beta(2)-adrenergic receptors.

beta-Adrenergic receptors (beta-AR) are essential regulators of cardiovascular homeostasis. In addition to their prominent function in the heart, beta-AR are located on vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines. In this study, we have investigated in an isometric myograph different types of blood vessels from mice lacking beta(1)- and/or beta(2)-adrenergic receptor subtypes (beta(1)-KO, beta(2)-KO, beta(1)beta(2)-KO). In wild-type mice, isoproterenol induced relaxation of segments from thoracic aorta, carotid, femoral and pulmonary arteries, and portal vein. The relaxant effect of beta-receptor stimulation was absent in femoral and pulmonary arteries from beta(1)-KO mice. In aortic and carotid arteries and in portal veins, the vasodilating effect of isoproterenol was reduced in mice lacking beta(1)- or beta(2)-receptors. However, in these vessels the vasodilating effect was only abolished in double KO mice lacking both beta(1)- and beta(2)-receptors. Vessel relaxation induced by forskolin did not differ between wild-type and KO mice. Similar contributions of beta(1)- and beta(2)-receptors to isoproterenol-induced vasorelaxation were found when vessels from KO mice were compared with wild-type arteries in the presence of subtype-selective beta-receptor antagonists. These studies demonstrate that beta(1)-adrenergic receptors play a dominant role in the murine vascular system to mediate vasodilation. Surprisingly, beta(2)-receptors contribute to adrenergic vasodilation only in a few major blood vessels, suggesting that differential distribution of beta-adrenergic receptor subtypes may play an important role in redirection of tissue perfusion.

Animals↗
PubMed2001

Receptor-ligand interactions studied with homogeneous fluorescence-based assays suitable for miniaturized screening.

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and beta(2)-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1 microl without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Betacellulin↗
PubMed2001

Constitutive activity of the human beta(1)-adrenergic receptor in beta(1)-receptor transgenic mice.

We tested the hypothesis that the human beta(1)-adrenergic receptor displays constitutive activity and that beta-adrenergic antagonists differ in their ability to modulate this constitutive activity. Transfection of the cDNAs of the human beta(1)- and beta(2)-adrenergic receptors into COS-7 cells caused increases in basal cAMP that were proportional to the receptor levels, thus demonstrating constitutive activity for both subtypes. At comparable receptor levels, the increase in basal cAMP was about 5-fold higher for the beta(2)- than for the beta(1)-subtype. As a model for enhanced beta-adrenergic signaling at the whole-organ level, we used transgenic mice with heart-specific overexpression of the human beta(1)-adrenergic receptor. In this model, the beta(1)-adrenergic receptor displayed constitutive activity as evidenced by a higher spontaneous beating rate of isolated right atria from beta(1)-transgenic versus wild-type mice. This difference was abolished by the addition of CGP20712A, demonstrating inverse agonist properties of this compound. We then tested whether various beta-adrenergic antagonists currently in clinical use for the treatment of heart failure differ in their ability to modulate constitutive activity of the cardiac beta(1)-adrenergic receptor. The beta(1)-selective antagonists metoprolol and bisoprolol showed significant inverse agonist activity at the beta(1)-adrenergic receptor. Carvedilol behaved as a neutral antagonist and xamoterol displayed marked partial agonist activity. We conclude that the human beta(1)-adrenergic receptor displays constitutive activity that is considerably lower than that of the beta(2)-subtype. beta-Adrenergic antagonists currently in clinical use differ in their ability to exert inverse agonist activity at the human beta(1)-adrenergic receptor, which may contribute to their therapeutic effects.

Adrenergic Antagonists↗
PubMed2000

Paradoxical block of parathormone secretion is mediated by increased activity of G alpha subunits.

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G alpha subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G alpha subunits of heterotrimeric G-proteins.

Animals↗
PubMed2000

Acute changes of myocardial creatine kinase gene expression under beta-adrenergic stimulation.

Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.

Adrenergic beta-Agonists↗
PubMed2000

Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin.

G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC). Whereas PKC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms alpha, gamma, and delta. Two-dimensional peptide mapping of PKCalpha-phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulin-binding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PKC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2.

Animals↗
PubMed2000

Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

Animals↗