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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 20, 2005 as doi:10.1096/fj.05-4688fje. |
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,1
* Department of Kinesiology, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, Japan;
Department of Molecular Predictive Medicine and Sport Science, School of Medicine, Kyorin University, Mitaka, Tokyo, Japan
2 Correspondence: Department of Kinesiology, Graduate School of Science, Tokyo Metropolitan University, 1-1, Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan. E-mail: izawa{at}comp.metro-u.ac.jp
SPECIFIC AIMS
Acute exercise-induced increase in the density of ß-adrenergic receptor (ß-AR) on the cell surface has been shown to involve trafficking of ß-AR from the cytosol to the membranes in human lymphocytes and rat myocardium. However, the molecular mechanism underlying acute exercise-induced traffic of the ß-AR has not been established and it not known whether such adaptation of ß-AR to exercise occurs in other tissues that play a role in metabolic homeostasis during exercise. The purpose of this study was to confirm whether acute exercise induces the traffic of ß-ARs from cytosol to the plasma membranes in rat adipocytes and, second, if that is the case to establish the molecular mechanism underlying exercise-induced trafficking of ß-ARs.
PRINCIPAL FINDINGS
1. Exercise increases cell surface number of ß-AR
In intact adipocytes, the binding sites of [3H]CGP-12177, which is ß1/ß2-AR-antagonistic and ß3-AR-agonistic hydrophilic ligand, significantly increased at immediately (0 h) and 3 h after exercise but significantly decreased at 24 h after exercise. These data indicate that the total number of cell surface ß-AR significantly increased at 0 h and 3 h after exercise but decreased at 24 h after exercise.
2. Protein expressions of three subtypes of ß-AR after exercise
We next tested the protein expressions of three subtypes of ß-AR, ß1-, ß2-, or ß3-AR, in two subcellular fractions, the cytosol and the membrane-rich pellet fractions, by Western blot analysis. As shown in Fig. 1
A, B, no significant change in ß1-AR protein was found. The ß2-AR protein in the membrane fraction, however, increased to 
140% of the control value obtained from nonexercising rats at 0 h and 3 h after acute exercise, but that in the cytosol fraction decreased (Fig. 1C, D
). The ß2-AR protein at 24 h after exercise significantly decreased in the membrane fraction but not in the cytosol fraction. The ß3-AR protein in the membrane fraction increased, but that in cytosol fraction decreased at 3 h after exercise (Fig. 1E, F
). Thus, the immunoblot analysis data revealed that the increased number of cell surface ß-ARs at 0 h after exercise would be due to an alteration in the proportion between ß2-ARs at the membrane and in deeper compartments of the cells; the former increased, but the latter decreased.
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3. mRNA expressions of the ß2-AR gene after exercise
No significant change in the ß2-AR mRNA was found at 0 h and 3 h after exercise. However, ß2-AR mRNA decreased significantly at 24 h after exercise.
4. GRK-2 protein expression after exercise
G-protein-coupled receptor kinase (GRK)-2 protein in the membrane fraction decreased significantly at 0 h and 3 h after exercise but returned to the control level at 24 h after exercise. In contrast, in the cytosol fraction, no significant change was observed at any of the designated times after exercise.
5. Expressions of ß-arrestins, ß-arrestin-2 and ß2-AR protein complex, and ubiquitination of ß2-AR after exercise
As shown in Fig. 2
C, D, ß-arrestin-2 protein expression in the membrane fraction decreased significantly at 0 h and 3 h after exercise, but returned to the control level at 24 h after exercise. However, in the cytosol fraction, these changes were not observed at any of the designed times after exercise. No change in the expression of ß-arrestin-1 protein was observed (Fig. 2A, B
). The association of ß2-AR and ß-arrestin-2 was reduced at 0 h and 3 h after exercise but returned to the control level at 24 h after exercise (Fig. 2E, F
). The association of ubiquitin protein and ß2-AR also decreased significantly at 0 h and 3 h after exercise, whereas the level of its association returned to the control level at 24 h after exercise.
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6. Effect of an intraperitoneal injection of either lactacystin or propranolol on GRK-2 and ß-arrestin-2 proteins
The injection of lactacystin, a selective proteasome inhibitor, induced no change in either GRK-2 or ß-arrestin-2 protein at any of the designed times after exercise compared with vehicle-injected control rats who were not subjected to exercise. Moreover, the administration of propranolol, a ß-adrenergic receptor blocker, also resulted in the change in neither GRK-2 nor ß2-AR protein after exercise.
7. Intracellular cAMP accumulations
The cellular contents of cAMP in response to a ß2-AR specific agonist, salbutamol (1 mM), significantly increased at 0 h and 3 h after exercise as compared with the control but significantly decreased at 24 h after exercise. BRL 37344 (10 µM), a ß3-AR-specific agonist, had a greater effect on intracellular cAMP production. Its effect was significantly enhanced at 3 h after exercise. However, at 24 h after exercise, BRL 37344 failed to increase intracellular cAMP.
CONCLUSIONS AND SIGNIFICANCE
In the current study, evidence is provided that in rat adipocytes, an exercise-induced increase in cell surface number of ß-ARs was mainly due to an alteration in the proportion between ß2-ARs at the membrane and in deeper compartments of the cells, and that this phenomenon may be mediated via reductions in a multi-step event involving the coordinate interaction among proteins (GRK-2 and ß-arrestin-2) mediating ß2-AR trafficking, in which both the receptor-agonist interactions and ubiquitin-proteasome pathway may have a key role. The decreased protein expression of ß2-AR at 24 h, on the contrary, might be due to some change occurring at the translational levels.
A number of studies have identified distinct properties of each ß-AR subtype for its trafficking mediated by GRK and ß-arrestin. For example, the interaction of ß1-AR with ß-arrestin is weak compared with that of ß2-AR. ß3-AR lacks most of the serine/threonine residues that are phosphorylated by the GRK and by PKA, and does not bind to ß-arrestin. Therefore, these distinct properties will explain the fact that ß2-AR was most efficiently redistributed under the condition in which exercise altered both GRK-2 and ß-arrestin-2 proteins.
GRK-mediated phosphorylation promotes the binding of ß-arrestin proteins, and the ubiquitination of ß2-AR, thereafter, occurs as a consequence of ß-arrestins binding to E3 ubiquitin ligase Mdm2. In line with this, significant reductions in both GRK-2 and ß-arrestin-2 protein and a simultaneous decline in the ubiquitination of ß2-AR were found in the membrane fraction at least up to 3 h after exercise. These data suggest that the internalization of some ß2-ARs would be hampered as a consequence of the blunted interaction among ß2-AR, GRK-2, and ß-arrestin-2. No alteration in the expression of ß2-AR mRNA at 0 h after exercise suggests that the increased ß2-AR protein in the membrane fraction did not involve a newly synthesized protein. Thus, the efficiency of the catecholamine-induced, GRK/ß-arrestin-2-mediated internalization of ß2-AR would be blunted during exercise. Given that the recycling efficiency of ß2-AR was maintained, the ß2-AR protein might be reduced in cytosolic fraction but increased in the membrane fraction. The chain of these events might result in an accumulation of ß2-AR on the cell surface. The administration of either propranolol or lactacystin completely blocked exercise-induced alterations in GRK-2 and b-arrestin-2, suggesting that both the receptor-agonist interactions and ubiquitin-proteasome pathway have a key role in exercise-induced ß2-AR trafficking.
The down-regulations of both GRK-2 and ß-arrestin-2 proteins last. Therefore, the reduced coupling efficiency of ß2-AR with GRK-2/ ß-arrestin-2 might also last at least up to 3 h after exercise; therefore, the up-regulated ß2-AR could be kept on the cell surface for at least 3 h after exercise. In contrast, at 24 h after exercise, the levels of GRK-2 and ß-arrestin-2 proteins and the ß2-AR/ß-arrestin-2 complex and the ubiquitination of ß2-AR returned to their respective control levels. However, the significant reduction in ß2-AR mRNA expression might lead to the reductions in ß2-AR protein and the cell surface number of ß-AR. The possible mechanisms behind the phenomena found in the study are depicted in Fig. 3
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The identity of the signaling molecules and their levels expressed in the plasma membrane are critical to determine the physiological and biochemical responses. The obtained data, therefore, may shed light on understanding how metabolic and mechanical responses of several tissues to catecholamines are regulated during exercise. The data obtained from our in vivo model might also be a good guide to the studies elucidating a mechanism behind ß-AR trafficking in the research fields other than exercise physiology and biochemistry.
FOOTNOTES
1 Current address: Department of Environmental and Preventive Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4688fje;
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