Regulation of G-alpha(i) palmitoylation.

Format:

Book

Thesis/Dissertation

Author/Creator:

Chen, Catherine A.

Contributor:

University of Pennsylvania.

Language:

English

Subjects (All):

Biochemistry.

Pharmacology.

0419.

0487.

Local Subjects:

0419.

0487.

Physical Description:

161 pages

Contained In:

Dissertation Abstracts International 61-10B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

Heterotrimeric GTP-binding regulatory proteins (G proteins), consisting of alpha, beta, and gamma subunits, transduce signals from G protein-coupled receptors at the cell surface to effector molecules in the intracellular milieu. Nearly all alpha subunits are palmitoylated at cysteine residues near the N-terminus. A regulated cycle of palmitoylation could provide a mechanism for modulating G protein signaling by affecting protein interactions and subcellular localization of the subunit. In the present studies we utilized both [ 3H]pahnitate incorporation and pulse-chase techniques to address the dynamics of alphai palmitoylation in the plasma membrane and caveolin-enriched microdomains of intact Chinese hamster ovary (CHO) cells.

For plasma membrane, both techniques demonstrated a significant dose- and time-dependent change in [3H]palmitate labeling of alpha i upon activation of stably expressed 5-hydroxytryptamine-1A (5-HT 1A) receptors by the agonist (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (DPAT), with an EC50 of ∼10 nM. For the incorporation assay, DPAT elicited an increase in labeling at the earliest time point measured. For the pulse-chase assay, DPAT promoted a loss of radiolabel almost equally as fast. These data demonstrate that the exchange of palmitate on alpha i is increased upon stimulation of 5-HT1A receptors through the combined processes of depalmitoylation and palmitoylation. These results provide the basis for extending the concept of regulated exchange of palmitate beyond Gs and provide a framework for exploring the specific functional attributes of the palmitoylated and depalmitoylated forms of the alpha subunit.

For caveolin-enriched microdomains, both techniques demonstrated a significant change in [3H]palmitate labeling of alphai upon activation of stably expressed 5-HT1A receptors by the agonist DPAT. For alphai immunoprecipitated from caveolin-enriched membranes prepared by sucrose gradient centrifugation, DPAT elicited an increase in labeling as measured by the incorporation assay, and promoted a loss of radiolabel as measured by the pulse-chase assay. For alphai co-immunoprecipitating with caveolin, DPAT also promoted an increase in labeling as measured by the incorporation assay. Taken together these data suggest that in caveolin-enriched microdomains, the exchange of palmitate on alphai is increased upon stimulation of the 5-HT1A receptor. These results suggest that receptor·G protein coupling may occur in caveolae and provide further support for examining caveolae as signaling centers.

Notes:

Source: Dissertation Abstracts International, Volume: 61-10, Section: B, page: 5249.

Supervisor: David R. Manning.

Thesis (Ph.D.)--University of Pennsylvania, 2000.

Local Notes:

School code: 0175.

ISBN:

9780599966277

Access Restriction:

Restricted for use by site license.