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J. Biol. Chem., Vol. 282, Issue 45, 33064-33075, November 9, 2007

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Unique Hydrophobic Extension of the RGS2 Amphipathic Helix Domain Imparts Increased Plasma Membrane Binding and Function Relative to Other RGS R4/B Subfamily Members^*

Steven Gu, Janet He, Wing-Ting Ho, Suneela Ramineni, David M. Thal^¶, Ramanathan Natesh¹, John J. G. Tesmer^¶, John R. Hepler, and Scott P. Heximer²

From the Department of Physiology, Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario M5S 1A8, Canada, the Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30329, and the ^¶Chemical Biology Program and the Department of Pharmacology, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109

RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGS-box domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated G_q. Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or G_q. Together, these data indicate that novel features within the RGS2 amphipathic helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.

Received for publication, March 28, 2007 , and in revised form, September 6, 2007.

^* This work received technical and core facility support from the Cell Biology of Atherosclerosis Program, supported by Heart and Stroke Foundation of Canada Grant PRG 5738. This work was also supported by Heart and Stroke Foundation of Canada Grant NA 5291 (to S. P. H.), the Canadian Institute for Health Research Canada Research Chairs Program (to S. P. H.), and National Institutes of Health Grants R01s-NS37112 and GM61847 (to J. R. H.) and R01s-HL071818 and HL086865 (to J. J. G. T.). 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 Figs. 1 and 2.

¹ Recipient of short term fellowship support from the International Human Frontier Science Program Organization.

² The Canada Research Chair in Cardiovascular Physiology. To whom all correspondence should be addressed: Dept. of Physiology, Heart and Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-6048; Fax: 416-978-4373; E-mail: scott.heximer{at}utoronto.ca.

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