Heterotrimeric G protein subunits are located on rat liver endosomes.
BACKGROUND
Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors.


RESULTS
By Western blotting Gsalpha, Gialpha1,2, Gialpha3 and Gbeta were enriched in both canalicular (CM) and basolateral (BLM) membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC) > compartment for uncoupling of receptor and ligand (CURL) > multivesicular bodies (MVB) >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC) was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsalpha, Gialpha3 and Gbeta corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers.


CONCLUSIONS
We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s) of internalized insulin receptors.
Van Dyke
Rebecca W
RW
Dept of Internal Medicine, University of Michigan School of Medicine and Veterans Administration Hospital, Ann Arbor, MI 48105, USA. wynne@umich.edu
D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002772:Cholera Toxin / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000737:chemistry*; D022781:Hepatocytes / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D020962:Heterotrimeric GTP-Binding Proteins / Q000032:analysis*; D008247:Lysosomes / Q000737:chemistry / Q000648:ultrastructure; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D015398:Signal Transduction
D043562:Receptors, G-Protein-Coupled; D002772:Cholera Toxin; D020962:Heterotrimeric GTP-Binding Proteins; D000262:Adenylyl Cyclases