Abstract
The Gs protein subunit (αs) sequences conserved in non-myristoylated α subunits (TENIR, residues 369-373) or critical for adenylyl cyclase interaction were investigated as possible sites required for membrane localization. Substitutions were created by site-directed mutagenesis in which the TENIR residues were deleted from αs or added to the soluble, non-myristoylated αil. After transfection, COS cells were separated by centrifugation into particulate and soluble fractions. Immunoblots showed that these substitutions did not change the localization: αs ± TENIR in the particulate fraction, non-myristoylated αil ± TENIR in the soluble fraction. The constitutively active αi/αs chimera (CH4A), containing four regions of αs sufficient for adenylyl cyclase activation, was mutated to prevent myristoylation (GA-CH4A). Immunoblots of transfected COS cell fractions showed CH4A in the particulate and GA-CH4A in the soluble fraction. While these regions did not lead to memebrane localization, the soluble GA-CH4A could activate adenylyl cyclase in the intact cell and after reconstitution with cyc- membranes.
Original language | English (US) |
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Pages (from-to) | 25-33 |
Number of pages | 9 |
Journal | Cellular Signalling |
Volume | 6 |
Issue number | 1 |
DOIs | |
State | Published - Jan 1994 |
Externally published | Yes |
Keywords
- COS cell transfection
- G protein
- adenylyl cyclase
- membrane attachment
- protein domains
- protein targeting
- site-directed mutagenesis
ASJC Scopus subject areas
- Cell Biology