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J. Biol. Chem., Vol. 282, Issue 8, 5726-5735, February 23, 2007

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Analysis of Lipolytic Protein Trafficking and Interactions in Adipocytes^*

James G. Granneman¹, Hsiao-Ping H. Moore², Rachel L. Granneman, Andrew S. Greenberg, Martin S. Obin, and Zhengxian Zhu

From the Department of Psychiatry and Behavioral Neurosciences, Center for Integrative Metabolic and Endocrine Research, Wayne State University School of Medicine, Detroit, Michigan 48201 and the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.

Received for publication, November 14, 2006 , and in revised form, December 12, 2006.

^* This work was supported in part by grants from the National Institutes of Health (DK 62292), the American Diabetes Association, the Fund for Medical Research and Education at WSU, and the Joseph Young Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Videos S1-S8.

² Present address: College of Arts and Sciences, Lawrence Technological University, Southfield, MI, 48075.

¹ To whom correspondence should be addressed: Dept. of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, 550 East Canfield, Detroit, MI 48201. Tel.: 313-577-5629; Fax: 313-577-9469; E-mail: jgranne{at}med.wayne.edu.

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