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 Natesh, John J G Tesmer, John R. Hepler, Scott P. Heximer

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Abstract

RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2is amuchmore 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 Gαq. 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 Gαq. Together, these data indicate that novel features within the RGS2 amphipathic α helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.

Original languageEnglish
Pages (from-to)33064-33075
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number45
DOIs
Publication statusPublished - 9 Nov 2007
Externally publishedYes

Cite this

Gu, Steven ; He, Janet ; Ho, Wing Ting ; Ramineni, Suneela ; Thal, David M. ; Natesh, Ramanathan ; Tesmer, John J G ; Hepler, John R. ; Heximer, Scott P. / Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 45. pp. 33064-33075.
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abstract = "RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2is amuchmore 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 Gαq. 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 Gαq. Together, these data indicate that novel features within the RGS2 amphipathic α helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.",
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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. / Gu, Steven; He, Janet; Ho, Wing Ting; Ramineni, Suneela; Thal, David M.; Natesh, Ramanathan; Tesmer, John J G; Hepler, John R.; Heximer, Scott P.

In: Journal of Biological Chemistry, Vol. 282, No. 45, 09.11.2007, p. 33064-33075.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members

AU - Gu, Steven

AU - He, Janet

AU - Ho, Wing Ting

AU - Ramineni, Suneela

AU - Thal, David M.

AU - Natesh, Ramanathan

AU - Tesmer, John J G

AU - Hepler, John R.

AU - Heximer, Scott P.

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N2 - RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2is amuchmore 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 Gαq. 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 Gαq. Together, these data indicate that novel features within the RGS2 amphipathic α helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.

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