Abstract
Regions of basic amino acids in proteins can promote membrane localization through electrostatic interactions with negatively charged membrane lipid head groups. Previous work showed that the heterotrimeric G protein subunit αq contains a polybasic region in its N terminus that contributes to plasma membrane localization. Here, the role of the N-terminal polybasic region of αq in signaling was addressed. For αq mutants, loss of plasma membrane localization correlated with loss of signaling function, as measured by the ability to couple activated G protein-coupled receptors (GPCRs) to stimulation of inositol phosphate production. However, recovery of plasma membrane localization of αq polybasic mutants by introduction of a site for myristoylation or by coexpression of βγ failed to recover signaling, suggesting a role for N-terminal basic amino acids of αq beyond simple plasma membrane localization. It is noteworthy that an αq4Q mutant, containing glutamine substitutions at arginines 27, 30, 31, and 34, was identified that failed to mediate signaling yet retained plasma membrane localization. Although αq4Q failed to couple activated receptors to inositol phosphate production, it was able to bind βγ, bind RGS4 in an activation-dependent manner, stimulate inositol phosphate production in a receptor-independent manner, and productively interact with a GPCR in isolated membranes. It is noteworthy that αq4Q showed a differing localization to plasma membrane nanodomains compared with wild-type αq. Thus, basic amino acids in the N terminus of αq can affect its lateral segregation on plasma membranes, and changes in such lateral segregation may be responsible for the observed signaling defects of αq4Q.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants GM56444, GM066717, GM066717]; the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK07705]; and the Swiss National Science Foundation Fellowship [Grant PA00A-111446].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.066340.
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ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- PM
- plasma membrane
- HEK
- human embryonic kidney
- HA
- hemagglutinin
- PAGE
- polyacrylamide gel electrophoresis
- RGS
- regulator of G protein signaling
- FBS
- fetal bovine serum
- PMSF
- phenylmethylsulfonyl fluoride
- MOI
- multiplicity of infection
- FRET
- fluorescence resonance energy transfer
- PLC-β
- phospholipase Cβ
- mCFP
- monomeric cyan fluorescent protein
- M3R
- M3 receptor
- Sf9
- Spodoptera frugiperda
- α2AR
- α2 adrenergic receptor
- [35S]GTPγS
- guanosine 5′-O-(3-[35S]thio)triphosphate
- UK14304
- 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline
- IP
- inositol phosphate
- U46619
- (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2 -oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid
- NTA
- nitrilotriacetic acid.
- Received May 12, 2010.
- Accepted July 21, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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