Functional Importance of the Amino Terminus of G
(*)
- John R. Hepler(1),
- Gloria H. Biddlecome(1),
- Christiane Kleuss(1),
- Laura A. Camp(2),
- Sandra L. Hofmann(2),
- Elliott M. Ross(1) and
- Alfred G. Gilman(1)(§)
- From the (1)Departments of Pharmacology and
- (2)Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235
- § To whom correspondence should be addressed. Tel.: 214-648-2370; Fax: 214-648-8812.
Abstract
G
is palmitoylated at residues Cys9 and Cys
. Removal of palmitate from purified G
with palmitoylthioesterase in vitro failed to alter interactions of G
with phospholipase C-β1, the G protein β
subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated G
(removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation
of G
. However, truncated G
and the single Cys mutants activated phospholipase C-β1 normally, while the double Cys mutants were poor activators. Loss
of both Cys residues impaired but did not abolish interaction of G
with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293
or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of
nonpalmitoylated G
proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction
of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34
of G
caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at
the amino terminus of G
are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character
to the protein distinct from that contributed by palmitate.
Footnotes
-
↵* This work was supported by National Institutes of Health Grant GM34497, American Cancer Society Grant BE30-O, the Lucille P. Markey Charitable Trust, and the Raymond and Ellen Willie Chair of Molecular Neuropharmacology (to A. G. G.), American Heart Association, Texas Affiliate Award 94G-112 (to J. R. H.), National Institutes of Health Grant GM30355 and Robert A. Welch Foundation Grant I-0982 (to E. M. R.), and National Institutes of Health Grant CA61823 (to S. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- G proteins
-
heterotrimeric guanine nucleotide-binding regulatory proteins
- G
-
the α subunit of a G protein
- G
-
the β
subunit complex of a G protein
- GTP
S -
guanosine 5′-3-O-(thio)triphosphate
- HPLC
-
high performance liquid chromatography
- BSA
-
bovine serum albumin
- PAGE
-
polyacrylamide gel electrophoresis
- NTA
-
nitrilotriacetic acid.
-
↵2G. H. Biddlecome, G. Berstein, and E. M. Ross, manuscript in preparation.
-
↵3The label from [3H]palmitate associated with cytosolic G
is myristate, not palmitate(10). In the experiment shown in Fig. 1A, G
is visualized as a pair of proteins with apparent molecular masses of 42 and 43 kDa. This is the result of unexpectedly efficient
reading of the altered polyhedron initiator codon contained upstream of the inserted G
sequence in the original pVL1393 expression vector(29). This altered initiator codon was placed out of frame with the G
sequence in all subsequent experiments.
-
↵4The m1 muscarinic receptor can stimulate nucleotide exchange on G
in the absence of agonist, albeit at a slower rate than that observed in the presence of an agonist such as carbachol(35).
-
↵5Much longer exposures reveal that a very small amount of label (<2% of wild type) is incorporated into truncated G
.
-
↵6Prior studies (G. H. Biddlecome, G. Berstein, and E. M. Ross, unpublished results) demonstrated that addition of a hexahistidine tag at the carboxyl terminus of G
decreased the capacity of phospholipase C-β1 to stimulate steady-state GTP hydrolysis (i.e. GAP effect) by impairing the interaction of G
with the m1 receptor that is necessary for rapid GDP/GTP exchange. Present studies revealed that nontagged and hexahistadine-tagged
G
shared similar rates of agonist-stimulated GTP
S binding, whereas the tagged protein had lower rates of atropine- (basal) and carbachol-stimulated steady-state GTP hydrolysis,
accounting for the higher relative level of stimulation by carbachol (Fig. 5; Table 1).
-
↵7When subjected to TX-114 phase separation analysis, purified G
-short partitioned predominantly into the detergent phase. Treatment of G
-short with palmitoylthioesterase failed to alter this pattern. These results provide further evidence that G
-short is not palmitoylated.
-
↵8P. Sternweis and C. Slaughter, personal communication.
-
- Received August 31, 1995.
- Revision received October 20, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
