Biomaterial Database

Human muscarinic cholinergic receptor Hm1 internalizes via clathrin-coated vesicles. 1996

L M Tolbert, and J Lameh

Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143-0446, USA.

The mechanism by which muscarinic receptors internalize upon agonist exposure is poorly understood. To determine the endocytic pathways responsible for muscarinic receptor internalization, we have stably transfected human embryonic kidney (HEK 293) cells with the Hm1 (human muscarinic subtype 1) receptor tagged at the amino terminus with the epitope EYMPME. The subcellular location of the receptor was visualized by immunofluorescence confocal microscopy and quantified with the use of binding studies. The receptor redistributed into intracellular compartments following agonist treatment. This process was reversible upon removal of agonist and inhibited by antagonist. Acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, inhibited carbachol-stimulated internalization. Phorbol 12-myristate 13-acetate, on the other hand, which inhibits caveolae-mediated endocytosis, had no effect on carbachol-induced endocytosis. Double-labeling confocal microscopy was used to characterize the intracellular vesicles containing Hm1 receptor following agonist treatment. The Hm1 receptor was shown to be colocalized with clathrin and alpha-adaptin, a subunit of the AP2 adaptor protein which links endocytosed proteins with clathrin in the intracellular vesicles. In addition, endosomes containing Hm1 also contained the transferrin receptor, which internalizes via clathrin-coated vesicles. In contrast, caveolin, the protein that comprises caveolae, did not colocalize with Hm1 in intracellular vesicles following agonist treatment, indicating that caveolae are not involved in the agonist-induced internalization of Hm1. These results indicate that agonist-induced internalization of the Hm1 receptor occurs via clathrin-coated vesicles in HEK cells.

UI	MeSH Term	Description	Entries
D007150	Immunohistochemistry	Histochemical localization of immunoreactive substances using labeled antibodies as reagents.	Immunocytochemistry,Immunogold Techniques,Immunogold-Silver Techniques,Immunohistocytochemistry,Immunolabeling Techniques,Immunogold Technics,Immunogold-Silver Technics,Immunolabeling Technics,Immunogold Silver Technics,Immunogold Silver Techniques,Immunogold Technic,Immunogold Technique,Immunogold-Silver Technic,Immunogold-Silver Technique,Immunolabeling Technic,Immunolabeling Technique,Technic, Immunogold,Technic, Immunogold-Silver,Technic, Immunolabeling,Technics, Immunogold,Technics, Immunogold-Silver,Technics, Immunolabeling,Technique, Immunogold,Technique, Immunogold-Silver,Technique, Immunolabeling,Techniques, Immunogold,Techniques, Immunogold-Silver,Techniques, Immunolabeling
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011976	Receptors, Muscarinic	One of the two major classes of cholinergic receptors. Muscarinic receptors were originally defined by their preference for MUSCARINE over NICOTINE. There are several subtypes (usually M1, M2, M3....) that are characterized by their cellular actions, pharmacology, and molecular biology.	Muscarinic Acetylcholine Receptors,Muscarinic Receptors,Muscarinic Acetylcholine Receptor,Muscarinic Receptor,Acetylcholine Receptor, Muscarinic,Acetylcholine Receptors, Muscarinic,Receptor, Muscarinic,Receptor, Muscarinic Acetylcholine,Receptors, Muscarinic Acetylcholine
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002217	Carbachol	A slowly hydrolyzed CHOLINERGIC AGONIST that acts at both MUSCARINIC RECEPTORS and NICOTINIC RECEPTORS.	Carbamylcholine,Carbacholine,Carbamann,Carbamoylcholine,Carbastat,Carbocholine,Carboptic,Doryl,Isopto Carbachol,Jestryl,Miostat,Carbachol, Isopto
D002460	Cell Line	Established cell cultures that have the potential to propagate indefinitely.	Cell Lines,Line, Cell,Lines, Cell
D002966	Clathrin	The main structural coat protein of COATED VESICLES which play a key role in the intracellular transport between membranous organelles. Each molecule of clathrin consists of three light chains (CLATHRIN LIGHT CHAINS) and three heavy chains (CLATHRIN HEAVY CHAINS) that form a structure called a triskelion. Clathrin also interacts with cytoskeletal proteins.
D003034	Coated Pits, Cell-Membrane	Specialized regions of the cell membrane composed of pits coated with a bristle covering made of the protein CLATHRIN. These pits are the entry route for macromolecules bound by cell surface receptors. The pits are then internalized into the cytoplasm to form the COATED VESICLES.	Bristle-Coated Pits,Cell-Membrane Coated Pits,Bristle Coated Pits,Bristle-Coated Pit,Cell Membrane Coated Pits,Cell-Membrane Coated Pit,Coated Pit, Cell-Membrane,Coated Pits, Cell Membrane,Pit, Bristle-Coated,Pit, Cell-Membrane Coated,Pits, Bristle-Coated,Pits, Cell-Membrane Coated
D004705	Endocytosis	Cellular uptake of extracellular materials within membrane-limited vacuoles or microvesicles. ENDOSOMES play a central role in endocytosis.	Endocytoses