title: Angiotensin II receptor endocytosis involves two distinct regions of the cytoplasmic tail. A role for residues on the hydrophobic face of a putative amphipathic helix.
abstract: Following agonist stimulation, many receptors are rapidly internalized from the plasma membrane via a mechanism which presumably involves recognition motifs within the cytoplasmic domains of the receptor. We have previously demonstrated (Thomas, W. G., Thekkumkara, T. J., Motel, T. J., and Baker, K. M. (1995) J. Biol. Chem. 270, 207-213) that truncation of the angiotensin II (AT1A) receptor, to remove 45 amino acids from the cytoplasmic tail, markedly reduced agonist stimulated receptor endocytosis. In the present study, we have stably and transiently expressed wild type and carboxyl terminus mutated AT1A receptors in Chinese hamster ovary cells to identify regions and specific amino acids important for this process. Wild type AT1A receptors rapidly internalized (t1/2 = 2.5 min; Ymax = 76.4%) after AII stimulation. Using AT1A receptor mutants, truncated and deleted at the carboxyl terminus, two distinct regions important for internalization were identified: one membrane proximal site between residues 315-329 and another distal to Lys333, within the terminal 26 amino acids. Point mutations (Y302A, Y312A, L316F, Y319A, and K325A) were performed to identify residues contributing to the membrane proximal site. Mutation of Y302A, Y312A, and K325A had little effect on the rate (t1/2 = 4.3, 2.8, and 2.8 min) and maximal amount (Ymax = 81.7, 67.8, and 73.5%) of AII induced internalization. In contrast, L316F and Y319A mutations displayed an approximately 2.5-fold reduction in rate (t1/2 = 6.1 and 6.2 min) and L316F a decreased maximal level (Ymax = 38.1 and 71.4%, respectively) compared to wild type. Interestingly, Leu316 and Tyr319 are closely aligned within the hydrophobic aspect of a putative amphipathic helix, possibly representing an internalization motif for the AT1A receptor. We conclude that the AT1A receptor does not use NPXXY (NPLFY302) motif, first described for the beta 2-adrenergic receptor, to mediate agonist stimulated endocytosis. Rather, two distinct regions of the carboxyl terminus are utilized: one involving hydrophobic and aromatic residues on a putative alpha-helix and another serine/threonine-rich domain.
authors.affiliation: Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.
authors.forename: W G
authors.initials: WG
authors.lastname: Thomas
authors.forename: K M
authors.initials: KM
authors.lastname: Baker
authors.forename: T J
authors.initials: TJ
authors.lastname: Motel
authors.forename: T J
authors.initials: TJ
authors.lastname: Thekkumkara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004705:Endocytosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D017931:DNA Primers; D011945:Receptors, Angiotensin