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*
Department of Pediatric Immunology, Wilhelmina Children’s Hospital of the University Medical Center Utrecht, Utrecht, The Netherlands;
Department of Clinical Immunology, Hannover Medical School, Hannover, Germany; and
Institut für Medizinische Psychologie, Essen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenergic agonists. The responsiveness of these GPCRs
is turned off by the family of G protein-coupled receptor kinases
(GRK1–6). These kinases act by phosphorylating the GPCR in an
agonist-dependent manner, resulting in homologous desensitization of
the receptor. Although GRKs are widely expressed throughout the body,
leukocytes express relatively high levels of GRKs, in particular GRK2,
-3, and -6. We investigated whether in vivo the inflammatory disease
adjuvant arthritis (AA) induces changes in GRK expression and function
in the immune system. In addition, we analyzed whether the systemic
effects of AA also involve changes in GRKs in nonimmune organs. At the
peak of the inflammatory process, we observed a profound
down-regulation of GRK2, -3, and -6 in splenocytes and mesenteric lymph
node cells from AA rats. Interestingly, no changes in GRK were observed
in thymocytes and in nonimmune organs such as heart and pituitary.
During the remission phase of AA, GRK levels in spleen and mesenteric
lymph nodes are returning to baseline levels. The decrease in GRK2 at
the peak of AA is restricted to CD45RA+ B cells and
CD4+ T cells, and was not observed in CD8+ T
cells. In conclusion, we demonstrate in this study, for the first time,
that an inflammatory process in vivo induces a tissue-specific
down-regulation of GRKs in the immune system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenergic receptor (2, 3, 4, 5, 6, 7). The coupling efficiency of GPCRs is tightly regulated. Recently, it has been shown that immune activation induces expression of a class of proteins called regulators of G protein signaling that attenuate G protein signaling routes (8). Another important family of proteins involved in regulation of GPCR signaling is the family of GPCR kinases (GRKs). GRKs are a family of kinases consisting of six known subtypes (GRK1–6) that play a crucial role in agonist-induced desensitization of GPCRs. Cells of the immune system express GRK2, -3, -5, and -6, of which GRK2 is the most widely studied (9, 10, 11). Interestingly, the level of GRK2 in leukocytes is 4- to 5-fold the level found in other organs such as the heart (9).
GRKs are responsible for the rapid phosphorylation of activated
GPCRs that facilitates subsequent binding of regulatory proteins,
-arrestins. This process finally leads to homologous desensitization
via uncoupling of the receptor from the G protein (12).
GRKs and arrestins also play a key role in GPCR internalization,
dephosphorylation, and recycling, thus contributing to the extent of
both desensitization and resensitization of the receptors
(13). The extent of agonist-induced desensitization and
sequestration of GPCRs depends on the intracellular availability of
GRKs and -arrestins (14, 15). Changes in GRK expression
levels alter the efficacy of GPCR signal transduction in vivo and in
vitro (12, 16).
In patients with hypertension, an increase in GRK2 expression in PBMC has been reported (17). Apparently, the systemic effects of hypertension include changes in GRKs in immune cells. We have recently demonstrated that the chronic inflammatory disease rheumatoid arthritis (RA) is associated with low levels of GRKs in PBMC of these patients (18). In brain tissue from opiate addicts and in cardiac tissue from patients with chronic heart failure or myocardial ischemia, increased GRK levels have been observed (19, 20, 21, 22). In addition, in a murine model of heart failure, the targeted cardiac overexpression of a GRK2 dominant-negative mutant can prevent the development of cardiomyopathy (23). These data suggest that changes in GRKs can contribute to the induction and/or the severity of diseases, in which GPCRs play a pivotal role. In the present study, we investigated in a rat model whether the immune-mediated disease adjuvant arthritis (AA) induces changes in GRK expression in immune and nonimmune organs. Furthermore, we tested whether changes in the levels of GRKs in the various organs are related to the severity of the disease. Specifically, we determined the level of GRK2, -3, and -6 before, at the onset, at the peak, and after recovery from the inflammatory process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inbred male Lewis rats of 6–8 wk of age were obtained from the University of Limburg (Maastricht, The Netherlands). AA was induced on day 0 by a single intradermal injection in the base of the tail with 100 µl of CFA. CFA was prepared by mixing 5 mg of heat-killed Mycobacterium tuberculosis (strain H37Ra), ground to a fine powder with 1 ml IFA. M. tuberculosis and IFA were both purchased from Difco (Detroit, MI). Rats were examined daily for clinical signs of arthritis by standard methodology (24).
Tissues and cells
On days 11, 18, 37, and 45 after arthritis induction, healthy and arthritic rats were decapitated and organs were collected. Pituitary and heart were frozen in liquid nitrogen and stored at -80°C. Immune organs (spleen, mesenteric lymph nodes (MLN), and thymus) were dispersed through filter chambers. Subsequently, splenocytes and MLN cells were isolated using Lympholite-rat (Cedarlane Laboratories, Hornby, Ontario, Canada) and Percoll (Pharmacia, Uppsala, Sweden) density gradients, respectively (25). CD4+ and CD8+ T cells and B cells were purified by magnetic cell sorting using MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and mAbs specific for CD4, CD8a, and CD45RA, respectively. Thymocytes were washed once in RPMI 1640 (Life Technologies, Grand Island, NY).
GRK activity
GRK enzymatic activity was assessed using light-dependent
phosphorylation of rhodopsin by cytosolic and membrane fractions from
splenocytes, as previously described (18). Samples were
electrophoresed on 10% SDS-PAGE. Phosphorylated rhodopsin was
visualized by autoradiography. Bands corresponding to rhodopsin (
38
kDa) were quantified via liquid scintillation spectroscopy.
GRK and arrestin expression
Cell lysates were obtained by lysis in ice-cold RIPA buffer (20 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10 mM EDTA, 2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AESBF), 20 µg/ml leupeptin, and 200 µg/ml benzamidine) for 30 min at 4°C. Proteins were separated by 10% SDS-PAGE (or 7.5% to assess GRK3) and analyzed for GRK and arrestin expression by immunoblotting as previously described (18). Plasma membrane fractions were used to assess GRK3, -5, and -6 since a significant amount of these kinases is associated with membranes (26, 27, 28). Immunodetection of myocardial levels of GRK2 was performed on detergent-solubilized extracts following immunoprecipitation (29).
GRK2, GRK5, and GRK6 were detected using a 1/200 dilution of rabbit
polyclonal Abs (Santa Cruz Biotechnology, Santa Cruz, CA). For GRK3,
the mAb C5/1, which recognizes an epitope common to GRK2 and -3, was
used at 1/5000 dilution (30). Expression of -arrestin-1
was determined using 1/500 dilution of a -arrestin-1 mouse mAb
(Transduction Laboratories, Lexington, KY). Immunoreactivity was
detected by ECL (Amersham, Buckinghamshire, U.K.). Autoradiographs were
scanned using a GS-700 Imaging Densitometer (Bio-Rad, Hercules,
CA).
Northern blot analysis
Total RNA was isolated using RNAzol-B (Campro Science,
Veenendaal, The Netherlands). A total of 10 µg of RNA/lane was
fractionated on a 1% agarose-formaldehyde gel and transferred to a
Hybond N+ membrane (Amersham). Northern blot
analysis was performed as described previously (18). GRK6
mRNA expression was determined on the same filter, after stripping with
0.5% SDS, using a random primed cDNA fragment (bp 1114–2030) of GRK6.
-actin mRNA expression was determined on the same membrane using a
random primed cDNA probe (1.8-kb human -actin cDNA; Clontech, Palo
Alto, CA).
Statistical analysis
Data are expressed as a mean value ± SE. All results were confirmed in at least two separate experiments. Specific measurements were compared using Student’s t test or one-way ANOVA, followed by Bonferroni’s analysis. Two-tailed p < 0.05 was considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first determined whether induction of AA results in an
alteration in GRK activity in splenocytes. AA was induced by injection
of rats with CFA, and disease scores were assessed. Rats did not show
any signs of arthritis until day 11 after inoculation. From day 11 to
day 18, there was a steep increase in the clinical score, reaching peak
values at days 18–20. On day 22, inflammatory signs began to decrease
until day 45, which was the last time point measured (Fig. 1
). Cytosolic fractions of splenocytes
were prepared from arthritic and control rats at day 18 after arthritis
induction and GRK enzymatic activity was determined. Rod outer segment
membranes were used as specific GRK substrate, resulting in a light
(agonist)-dependent phosphorylation of the correspondent 38-kDa band
(Fig. 2A, inset).
The results demonstrated clearly that GRK activity in cytosolic
fractions of splenocytes from AA rats was significantly decreased
compared with splenic cytosolic samples from control rats
(p < 0.001, Fig. 2A). Although GRKs are
essentially cytosolic proteins, a significant amount of kinase activity
is associated with the plasma membrane (31, 32). To rule
out the possibility that the observed decrease in GRK activity was
simply due to GRK translocation from cytosol to membrane fractions, we
also assessed membrane-associated kinase activity. We found a decrease
similar to that observed in cytosolic fractions (data not shown).
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We next determined whether the observed decrease in GRK activity
was associated with a decrease in GRK2 protein levels. Cell lysates
from splenocytes obtained from AA rats at day +18 after induction of
the disease as well as from naive control rats were examined by Western
blot analysis. The GRK2 Ab recognizes a protein of an apparent
molecular mass of 80 kDa that comigrates with rGRK2 protein (Fig. 2
B, inset). In splenocytes from AA rats,
immunodetectable GRK2 was significantly reduced when compared with
control rats (p < 0.001, Fig. 2B).
To determine which cell types are responsible for the observed decrease
in GRK2, we analyzed splenic CD4+ T cells,
CD8+ T cells, and CD45RA+ B
cell subpopulations isolated by magnetic cell sorting. Immunodetectable
levels of GRK2 were profoundly reduced in whole cell lysates from
CD45RA+ B cells (95% reduction,
p < 0.001) and CD4+ T cells
(80% reduction, p < 0.001). In contrast, no
changes were detected in CD8+ T cells (Fig. 2C).
GRK2 mRNA expression in spleen
Northern blot analysis of splenic RNA was performed to analyze
whether the down-regulation of GRK2 protein in AA is reflected at the
mRNA level. A cDNA probe derived from human GRK2 detected a major mRNA
species of 3.8 kb and a second minor band of 2.4 kb, known to be
present in rat cells (Fig. 2D). Our results demonstrate that
GRK2 mRNA levels in splenocytes from AA (n = 6) and
control rats (n = 6) were not significantly different
at day +18 after induction (GRK2: AA = 112 ± 5% of
controls) (Fig. 2D, left panel). Also on day +45
after AA induction, GRK2 mRNA levels in splenocytes from AA
(n = 5) and control rats (n = 5) did
not differ significantly (AA = 98 ± 3% of controls) (Fig. 2D, right panel).
GRK2 protein expression in other lymphoid organs
To assess whether the profound down-regulation of GRK2 observed in
cells obtained from spleen of AA rats was also present in other
lymphoid organs, we analyzed GRK2 expression in MLN cells and
thymocytes. In MLN cells of AA rats at day 18 after induction of
disease, GRK2 levels were reduced by 80% (p <
0.05, Fig. 3A). When
CD4+ T cells, CD8+ T cells,
and CD45RA+ B cells separated from MLN were
analyzed, the results were similar to those obtained from splenic
subpopulations: GRK2 levels were significantly decreased in
CD45RA+ B cells and CD4+ T
cells, whereas GRK2 levels in CD8+ T cells
remained unchanged (data not shown).
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A and inset B). GRK2 protein expression in heart and pituitary
To elucidate whether the down-regulation of GRK2 expression in AA
rats was restricted to the immune system, we analyzed GRK2 expression
in heart and pituitary. Myocardial levels of GRK2 protein were assessed
after immunoprecipitation (Fig. 3B and inset
C). Immunodetectable GRK2 in myocardial tissue from AA rats
on day 18 after induction of disease does not differ significantly from
GRK2 protein in myocardial tissue from control rats (Fig. 3B). Similarly, no significant differences in GRK2 protein
levels were detected in pituitary cytosolic (Fig. 3B and
inset D) and membrane fractions (data not shown) from AA and
control rats.
Time course of GRKs and -arrestin protein
expression in AA
Our next question was whether the change in GRK expression levels
is related to the course of the disease. Specifically, we examined
splenocytes and MLN cells obtained at disease onset (day 11), at the
peak of disease (day 18), and at days 37 and 45, when clinical signs of
inflammation had almost disappeared (Fig. 1). On days 11 and 18 after
AA induction, we observed a marked reduction in GRK2 protein levels (up
to 80–85%) in spleen and MLN cells. On day 37 after induction of
AA, GRK2 protein levels in spleen cells are still very low, whereas
GRK2 levels in MLN cells had returned to the level of control rats. At
day 45, GRK2 levels in splenocytes had increased significantly
(p < 0.01 vs day 18), but were still lower
than in control rats (50% decrease, p < 0.05)
(Fig. 4A). In thymocytes and
heart, no changes in GRK2 expression levels were detected at days 11,
37, and 45 (data not shown).
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45% (p < 0.05, Fig. 4B and inset) in splenocytes from AA rats on day
18 after induction and had returned to baseline level at day 37 (Fig. 4B). GRK5 was barely detectable in plasma membranes from
splenocytes of healthy and AA rats (data not shown). Immunodetectable
GRK6 in splenocytes was reduced profoundly by 80%
(p < 0.01) in AA rats on days 11 and 18 and
was back to normal on day 37 (Fig. 4C and inset).
Similarly to GRK2, the profound down-regulation of GRK6 expression
levels was not mirrored by a down-regulation at the mRNA level (GRK6:
AA = 103 ± 4% of controls at day +18).
Next, we investigated whether AA was associated with changes of the GRK
cofactor -arrestin-1. In splenocytes from AA rats on day 18 after
induction of the disease, we observed a moderate, but statistically
significant, increase in immunodetectable -arrestin-1 (Fig. 4D and inset) compared with controls (AA =
136 ± 5% of expression in controls on day 18, p
< 0.01). On day 45, -arrestin-1 levels in splenocytes had returned
to the levels of control rats (Fig. 4D and
inset). A similar time course for -arrestin-1 expression
was obtained in MLN cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenergic receptors by infusion of
isoproterenol for 14 days in rats induces changes in GRK2 levels in
heart as well as in PBMC (34). Moreover, spontaneously
hypertensive rats show a significant increase of immunodetectable GRK2
both in lymphocytes and vascular myocytes when compared with
normotensive rats (35). Based on these data, we expected
to find alterations in GRKs during arthritis not only in immune organs,
but also in nonimmune organs. However, although AA is a systemic
inflammatory disease, the changes in GRK levels were restricted to
secondary immune organs (Figs. 2 and 3). No change in GRKs was observed
in a primary immune organ (thymus) or in heart and pituitary of
arthritic rats (Fig. 3). Our results demonstrate that down-regulation
of GRKs only occurs at the site in which the immune response takes
place. In this respect it is of interest that only subsets that are
involved in arthritis, namely CD4+ T cells and B
cells (36, 37, 38), display decreased GRK2 levels. In
contrast, GRK2 remains unchanged in CD8+ T cells
that are not required for expression of the disease in animal models
(37, 38).
Down-regulation of GRK2 protein level in immune cells can be induced in
vitro by proinflammatory cytokines, e.g., IL-6 and IFN-
(18). Although receptors for proinflammatory cytokines are
present on many tissues, including thymus, pituitary, and heart
(39, 40, 41), and circulating levels of cytokines are
increased in arthritis (42), GRK levels in these nonimmune
organs are unaltered. Thus, if cytokines are responsible for GRK
decreases in immune organs of arthritic rats, one has to assume that
this effect of cytokines is limited to activated immune organs.
Previously, we reported that PBMC of humans with RA express lower
levels of GRK protein without changes in GRK mRNA (18). In
the present study, we demonstrate that a similar phenomenon occurs in
rat splenocytes during AA. The observation that mRNA levels for the
GRKs do not change during arthritis suggests that the down-regulation
of GRKs involves a process of posttranscriptional modification and/or
protein degradation. Nonspecific degradation of proteins can be
excluded since the level of -arrestin-1 does not decrease, but
rather increases during AA (Fig. 4D). Penela et al.
(43) recently described that
2-adrenergic receptor stimulation enhances
GRK2 degradation by the proteasome pathway. It may well be possible
that in arthritis mediators that signal through GPCR, for example
chemokines and catecholamines, enhance GRK2 degradation via proteasomes
through stimulation of their respective receptors (44, 45). In addition, we have preliminary evidence that activated
granulocytes can induce a down-regulation of GRK2 in PBMC of healthy
individuals via the production of oxygen radicals (M.S.L., A.K., L.
Scholtens, M. Roccio, M.S., R.E.S., and C.J.H., manuscript in
preparation). Oxygen radicals are produced in high amounts during
inflammatory processes by activated granulocytes and macrophages and
are potent activators of the proteasome pathway (46, 47).
Therefore, it is tempting to hypothesize that in activated immune
organs, oxygen radicals are responsible for local degradation of GRK2
protein. If this hypothesis is correct, the normal GRK2 levels in
heart, pituitary, and thymus of arthritic rats can be explained by the
absence of activated granulocytes or macrophages in these organs during
the disease process.
At the peak of AA in rats, we also observed a profound down-regulation
of GRK6 and, to a lesser extent, of GRK3 in spleen and MLN cells (Fig. 4). At the same time, the expression of -arrestin-1, another protein
involved in the desensitization/resensitization of GPCRs, is actually
increased (Fig. 4D). During the remission phase of
arthritis, GRK2 and GRK6 levels in spleen have partially or completely
returned to baseline levels. The fact that changes in GRK levels
develop after induction of arthritis in rats and return to normal
levels during the remission phase suggests that inflammatory activity
determines how much GRK will be present in immune cells. Our present
results in arthritic rats also suggest that in patients with RA, the
low GRK2 levels are the consequence of the disease process and do not
reflect preexisting low levels of GRK2 in these patients.
One of the functional effects of down-regulation of GRKs can be an
increased sensitivity of various GPCRs in vivo. We have demonstrated
that PBMC of RA patients, showing a 50% reduction in GRK2
expression and activity, are more sensitive to
2-adrenergic activation (18).
Furthermore, we have preliminary evidence that splenocytes of AA rats
have an enhanced response to triggering of the
PGE2 receptor, that is a substrate for GRKs. More
direct evidence for the physiological importance of GRK2 levels has
been obtained by expression of a GRK2 dominant-negative mutant in cells
that also express the chemokine receptor CCR2B. In those experiments,
it has been shown that the dominant-negative mutant prevents homologous
desensitization of the CCR2B receptor after exposure to the agonist
monocyte chemoattractant protein 1 (44). Conversely,
overexpression of GRK2 has been shown to enhance the desensitization of
the opiate receptor (48). Finally, in heterozygous
GRK2+/- knockout mice, the low GRK2 activity is
associated with increased in vivo contractile responses to the
2-adrenergic agonist isoproterenol (49, 50). Based on these data, we would like to propose that
down-regulation of GRKs during (chronic) inflammation may lead to
increased or sustained activation of G protein-coupled proinflammatory
receptors, e.g., chemokine receptors.
In conclusion, we demonstrated in this study, for the first time, that an inflammatory process in vivo induces tissue and cell type-specific down-regulation of GRKs in immune organs involved in the inflammatory response. Therefore, research aimed at specific regulation of GRKs may lead to new therapeutic strategies on the level of receptor regulation in chronic inflammatory diseases, such as rheumatoid arthritis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maria Stella Lombardi, Department of Pediatric Immunology, Room KC 03.068.0, Wilhelmina Children’s Hospital of the University Medical Center Utrecht, Lundlaan 6, 3584 EA-Utrecht, The Netherlands.
3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; AA, adjuvant arthritis; GRK, GPCR kinase; MLN, mesenteric lymph node; RA, rheumatoid arthritis.
Received for publication February 17, 2000. Accepted for publication November 9, 2000.
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References |
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  Y. Chen, H. Long, Z. Wu, X. Jiang, and L. Ma EGF Transregulates Opioid Receptors through EGFR-mediated GRK2 Phosphorylation and Activation Mol. Biol. Cell, July 1, 2008; 19(7): 2973 - 2983. [Abstract] [Full Text] [PDF]
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 S. Salim, K. M. Standifer, and D. C. Eikenburg Extracellular Signal-Regulated Kinase 1/2-Mediated Transcriptional Regulation of G-Protein-Coupled Receptor Kinase 3 Expression in Neuronal Cells J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 51 - 59. [Abstract] [Full Text] [PDF]
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 A. Vroon, C. J. Heijnen, and A. Kavelaars GRKs and arrestins: regulators of migration and inflammation J. Leukoc. Biol., December 1, 2006; 80(6): 1214 - 1221. [Abstract] [Full Text] [PDF]
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N. W. Kin and V. M. Sanders It takes nerve to tell T and B cells what to do J. Leukoc. Biol., June 1, 2006; 79(6): 1093 - 1104. [Abstract] [Full Text] [PDF]
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 N. D. Khoa, M. Postow, J. Danielsson, and B. N. Cronstein Tumor Necrosis Factor-{alpha} Prevents Desensitization of G{alpha}s-Coupled Receptors by Regulating GRK2 Association with the Plasma Membrane Mol. Pharmacol., April 1, 2006; 69(4): 1311 - 1319. [Abstract] [Full Text] [PDF]
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A. Vroon, C. J. Heijnen, M. S. Lombardi, P. M. Cobelens, F. Mayor Jr, M. G. Caron, and A. Kavelaars Reduced GRK2 level in T cells potentiates chemotaxis and signaling in response to CCL4 J. Leukoc. Biol., May 1, 2004; 75(5): 901 - 909. [Abstract] [Full Text] [PDF]
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 M. S. Lombardi, E. van den Tweel, A. Kavelaars, F. Groenendaal, F. van Bel, and C. J. Heijnen Hypoxia/Ischemia Modulates G Protein-Coupled Receptor Kinase 2 and {beta}-Arrestin-1 Levels in the Neonatal Rat Brain Stroke, April 1, 2004; 35(4): 981 - 986. [Abstract] [Full Text] [PDF]
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 A. Kavelaars, A. Vroon, R. P. Raatgever, A. M. Fong, R. T. Premont, D. D. Patel, R. J. Lefkowitz, and C. J. Heijnen Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6 J. Immunol., December 1, 2003; 171(11): 6128 - 6134. [Abstract] [Full Text] [PDF]
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T. A. Kohout and R. J. Lefkowitz Regulation of G Protein-Coupled Receptor Kinases and Arrestins During Receptor Desensitization Mol. Pharmacol., January 1, 2003; 63(1): 9 - 18. [Full Text] [PDF]
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P. M. Cobelens, A. Kavelaars, A. Vroon, M. Ringeling, R. van der Zee, W. van Eden, and C. J. Heijnen The {beta}2-Adrenergic Agonist Salbutamol Potentiates Oral Induction of Tolerance, Suppressing Adjuvant Arthritis and Antigen-Specific Immunity J. Immunol., November 1, 2002; 169(9): 5028 - 5035. [Abstract] [Full Text] [PDF]
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M. S. Lombardi, A. Kavelaars, P. Penela, E. J. Scholtens, M. Roccio, R. E. Schmidt, M. Schedlowski, F. Mayor Jr., and C. J. Heijnen Oxidative Stress Decreases G Protein-Coupled Receptor Kinase 2 in Lymphocytes via a Calpain-Dependent Mechanism Mol. Pharmacol., August 1, 2002; 62(2): 379 - 388. [Abstract] [Full Text] [PDF]
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 A. M. Fong, R. T. Premont, R. M. Richardson, Y.-R. A. Yu, R. J. Lefkowitz, and D. D. Patel Defective lymphocyte chemotaxis in beta -arrestin2- and GRK6-deficient mice PNAS, May 28, 2002; 99(11): 7478 - 7483. [Abstract] [Full Text] [PDF]
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