Angiotensin II Receptor Endocytosis Involves Two Distinct Regions of the Cytoplasmic Tail
1995; Elsevier BV; Volume: 270; Issue: 38 Linguagem: Inglês
10.1074/jbc.270.38.22153
ISSN1083-351X
AutoresWalter G. Thomas, Kenneth M. Baker, Thomas J. Motel, Thomas Thekkumkara,
Tópico(s)Ion channel regulation and function
ResumoFollowing 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 (t = 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 (t = 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 (t = 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 the NPXXY (NPLFY302) motif, first described for the β2-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 α-helix and another serine/threonine-rich domain. 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 (t = 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 (t = 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 (t = 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 the NPXXY (NPLFY302) motif, first described for the β2-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 α-helix and another serine/threonine-rich domain. INTRODUCTIONEndocytosis of cell surface receptors is a ubiquitous eukaryotic process. It permits the internalization of extracellular nutrients (e.g. cholesterol via low density lipoprotein receptors), serves to dampen cell responses by removing ligand-activated receptors from the cell surface, and mediates cellular resensitization by recycling functional receptors to the cell surface(1Goldstein J.L. Brown M.S. Anderson R.G.W. Russel D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1106) Google Scholar, 2Pearse B. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (535) Google Scholar, 3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (127) Google Scholar, 4Smythe E. Warren G. Eur. J. Biochem. 1991; 202: 689-699Crossref PubMed Scopus (138) Google Scholar, 5Vaux D. Trends Cell Biol. 1992; 2: 189-192Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 6Sorkin A. Water C.M. BioEssays. 1993; 15: 375-382Crossref PubMed Scopus (232) Google Scholar). Although some endocytosis occurs constitutively, the rate is increased dramatically following binding of many extracellular ligands to their cognitive transmembrane receptors. This process is homologous in that usually only receptors for the stimulating ligand are internalized, and of those, only receptors occupied by ligand are targeted. Homologous endocytosis predicts that receptors inherently contain within their structures and sequences determinants for internalization, and that these determinants are concealed or remain latent until agonist binding. Thus, it is generally assumed that within the cytoplasmic domains of receptors are one or more amino acid codes, or motifs, that are recognized by adaptor complexes which mediate the selective recruitment of receptor-ligand complexes into primarily clathrin-coated vesicles (3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (127) Google Scholar, 4Smythe E. Warren G. Eur. J. Biochem. 1991; 202: 689-699Crossref PubMed Scopus (138) Google Scholar, 5Vaux D. Trends Cell Biol. 1992; 2: 189-192Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 6Sorkin A. Water C.M. BioEssays. 1993; 15: 375-382Crossref PubMed Scopus (232) Google Scholar, 7Pley U. Parham P. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 431-464Crossref PubMed Scopus (68) Google Scholar, 8Robinson M. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (304) Google Scholar) or non-coated vesicles and caveolae (discussed in (9Roettger B.F. Rentsch R.U. Pinon D. Holicky E. Hadac E. Larkin J.M. Miller L.J. J. Cell Biol. 1995; 128: 1029-1041Crossref PubMed Scopus (202) Google Scholar)). Commonly, these motifs include aromatic (predominantly tyrosine) and hydrophobic amino acids, and while phosphorylation of these crucial tyrosines does not necessarily drive the internalization process, phosphorylation at other sites may initiate allosteric changes within the receptor responsible for unmasking endocytotic codes (10Sorkin A. McKinsey T. Shih W. Kirchhausen T. Carpenter G. J. Biol. Chem. 1995; 270: 619-625Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Opresko L.K. Chang C. Will B.H. Burke P.M. Gill G.N. Wiley H.S. J. Biol. Chem. 1995; 270: 4325-4333Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 12Nesterov A. Kurten R.C. Gill G.N. J. Biol. Chem. 1995; 270: 6320-6327Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).Angiotensin II (AII) 1The abbreviations used are: AIIangiotensin IIAT1,1A,1B,2angiotensin II receptor subtypesG-proteinguanyl nucleotide-binding proteinGCPR(s)G-protein coupled receptor(s)CHO-K1Chinese hamster ovary cellsG418geneticinMEMminimal essential mediumPCRpolymerase chain reaction. is a peptide hormone with important actions on blood pressure regulation, water and salt balance, neuromodulation, and cellular growth(13Robertson J.I.S. Nicholls M.G. The Renin-Angiotensin System. Vol. 1. Gower Medical Publishing, London1993Google Scholar). Two major types of AII receptors, termed AT1 and AT2, and two subtypes of the AT1 receptor, termed AT1A and AT1B, have been identified(14Sandberg K. Trends Endocrinol. Metab. 1994; 5: 28-35Abstract Full Text PDF PubMed Scopus (70) Google Scholar). These receptors are all members of the seven transmembrane, guanyl nucleotide-binding protein (G-protein)-coupled receptor (GPCR) superfamily, but the AT1A receptor, which contains 359 amino acids including approximately 54 amino acids as a carboxyl-terminal cytoplasmic tail, is the principal mediator of the biological actions of AII. AII receptors present on cells cultured from various tissues (15Crozat A. Penhoat A. Saez J.M. Endocrinology. 1986; 118: 2312-2318Crossref PubMed Scopus (92) Google Scholar, 16Griendling K.K. Delafontaine P. Rittenhouse S.E. Gimbrone Jr., M.A. Alexander R.W. J. Biol. Chem. 1987; 262: 14555-14562Abstract Full Text PDF PubMed Google Scholar, 17Ullian M.E. Linas S.E. J. Clin. Invest. 1989; 84: 840-846Crossref PubMed Scopus (113) Google Scholar, 18Hunyady L. Merelli F. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1991; 266: 2783-2788Abstract Full Text PDF PubMed Google Scholar, 19Anderson K.M. Murahashi T. Dostal D.E. Peach M.J. Am. J. Physiol. 1993; 284: C179-C188Crossref Google Scholar) and cloned AT1A and AT1B(20Conchon S. Monnot C. Teutsch B. Corvol P. Clauser E. FEBS Lett. 1994; 349: 365-370Crossref PubMed Scopus (93) Google Scholar, 21Hunyady L. Tian Y. Sandberg K. Balla T. Catt K.J. Kidney Int. 1994; 46: 1496-1498Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 22Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar, 23Hunyady L. Bor M. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 31378-31382Abstract Full Text PDF PubMed Google Scholar, 24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), but not AT2(23Hunyady L. Bor M. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 31378-31382Abstract Full Text PDF PubMed Google Scholar), receptors expressed in cell systems, rapidly internalize upon AII binding. Internalization of AII•receptor complexes is independent of G-protein coupling (20Conchon S. Monnot C. Teutsch B. Corvol P. Clauser E. FEBS Lett. 1994; 349: 365-370Crossref PubMed Scopus (93) Google Scholar, 21Hunyady L. Tian Y. Sandberg K. Balla T. Catt K.J. Kidney Int. 1994; 46: 1496-1498Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 22Hunyady L. Baukal A.J. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 24798-24804Abstract Full Text PDF PubMed Google Scholar) and occurs via a clathrin-dependent process(20Conchon S. Monnot C. Teutsch B. Corvol P. Clauser E. FEBS Lett. 1994; 349: 365-370Crossref PubMed Scopus (93) Google Scholar), which is the primary route for many, but not all, GPCRs. While the exact cellular processes and receptor motifs that control AT1A receptor endocytosis are unknown, the AT1A carboxyl terminus contains four candidate tyrosine residues (Tyr302, Tyr312, Tyr319, and Tyr326). Interestingly, Tyr302 in the motif NPXXY is analogous to Tyr326 shown to be important for β2-adrenergic receptor internalization(25Barak L.S. Tiberi M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar), but the contribution of Tyr302 to AT1A receptor endocytosis has not been reported. The serine/threonine-rich portion of the carboxyl terminus has been implicated in AT1A receptor internalization(23Hunyady L. Bor M. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 31378-31382Abstract Full Text PDF PubMed Google Scholar), and we recently demonstrated that truncation of the AT1A receptor to delete the carboxyl-terminal 45 amino acids markedly reduced agonist-mediated endocytosis(24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In the present study, we have stably and transiently expressed in CHO-K1 cells AT1A receptors containing truncation, deletion, and point mutations of the cytoplasmic tail and determined AII stimulated endocytosis. Our data implicate two separate domains within the carboxyl-terminal region of the AT1A receptor in endocytosis and identify key amino acids involved in this process.EXPERIMENTAL PROCEDURESReagents and Cell Culture Materials125I-AII (specific activity >2000 Ci/mmol) was purchased from DuPont NEN or Hazelton Laboratories (Vienna, VA). Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (Rockville, MD). The Escherichia coli strain XL1-blue and pBluescript II vector were obtained from Stratagene (La Jolla, CA). Sequenase 2.0 DNA sequencing kits were from U. S. Biochemical Corp. and the pRc/CMV vector was from Invitrogen (San Diego, CA). α-MEM, OPTI-MEM, fetal bovine serum, the antibiotic geneticin (G418), other cell culture additives and plasticware, and lipofectamine were obtained from Life Sciences. DNA modifying enzymes were from Promega (Madison, WI). All other chemicals were from Fisher or Sigma.Receptor Constructs and MutagenesisWe have previously reported the cloning and isolation of a genomic clone for the rat AT1A receptor (λ12), subcloning of the full-length receptor (coding for 359 amino acids) into pBluescript II (pB2/AT1A) and the subsequent incorporation of the receptor into the pRc/CMV eukaryotic expression vector (pRc2A/AT1A) (26Thekkumkara T.J. Du J. Dostal D.E. Motel T.J. Thomas W.G. Baker K.M. Mol. Cell. Biochem. 1995; 146: 79-89Crossref PubMed Scopus (42) Google Scholar). The construction of an AT1A receptor with a stop codon after Leu314, which codes for a receptor missing 45 amino acids from the carboxyl terminus, subcloning into pRc/CMV (pRc/TL314) and stable expression of this truncated receptor in CHO-K1 cells (TL314) has been previously described(24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).To truncate the AT1A receptor after Lys333, a 1126-base pair fragment was PCR amplified from the clone pB2/AT1A using a sense primer (5′-GTAAAGCTTAAGTGGATTTCG-3′) and antisense primer (5′-GGTAGAAAGCTTGCTCTATTTCGTAGAC-3′), incorporating HindIII restriction sites (underlined) and an antisense stop codon (italics) after Lys333. The amplified DNA was restriction digested with HindIII and subcloned directly into the pRc/CMV vector (pRc/TK333). Sequencing with the Sequenase 2.0 kit, as described previously(26Thekkumkara T.J. Du J. Dostal D.E. Motel T.J. Thomas W.G. Baker K.M. Mol. Cell. Biochem. 1995; 146: 79-89Crossref PubMed Scopus (42) Google Scholar), confirmed the correct orientation and the entire coding region. Deletion of 15 amino acids (Glu315 through Ser329) from the full cytoplasmic tail was constructed in two steps. First, a 1012-base pair fragment was PCR amplified from the clone pB2/AT1A using the same sense primer as above and an antisense primer (5′-TTTCAGGAGCTCGAGGAAATACTT-3′), incorporating a XhoI site (underlined). The PCR fragment, coding for amino acids 1-314, was restriction digested with HindIII and XhoI and subcloned into pBluescript II (pB/1-314). Second, a DNA fragment, coding for amino acids 330-359, was PCR amplified from pB2/AT1A using a sense primer (5′-TCCTGTCTACGAAAATGAGCACG-3′) and an antisense primer (5′-CTACAGTCTGATGGGCCCATTTTTCTGCTTAG-3′), incorporating an ApaI restriction site (underlined). pB1-314 was digested with XhoI, blunted using mung bean nuclease and a second restriction digest performed with ApaI. The second PCR product was digested with ApaI and ligated into the modified pB1-314. The integrity of the deletion mutant construct was confirmed by sequencing and then released by an ApaI/HindIII digest for cloning into pRc/CMV (pRc/Del315-329).Point mutations were introduced into the AT1A receptor using the PCR-based method of Ito et al.(27Ito W. Ishiguro H. Kurosawa Y. Gene (Amst.). 1991; 102: 67-70Crossref PubMed Scopus (261) Google Scholar). Tyrosines at positions 302, 312, and 319 and lysine at position 325 were individually mutated to alanine, and leucine at position 316 was mutated to phenylalanine, using the following primers (mutated bases are underlined): Y302A, 5′-AACCCTCTGTTCGCCGGCTTTCTG-3′; Y312A, 5′-GAAATTTAAAAAGGCTTTCCTCCAG-3′; L316F, 5′-GTATTTCCTCCAGTTCCTGAAATAT-3′; Y319A, 5′-CAGCTCCTGAAAGCTATTCCCCCAAAG-3′; K325A, 5′-CCCCCAAAGGCCGCGTCCCACTCA-3′.All mutated PCR fragments were cloned into pBluescript II and the entire coding region and the relevant mutation confirmed by sequencing. Mutated receptor sequences were restriction digested with HindIII and subcloned into pRc/CMV at the same site to yield the expression plasmids pRc/Y302A, pRc/Y312A, pRc/L316F, pRc/Y319A, and pRc/K325A.Stable and Transient TransfectionsCHO-K1 cells were maintained in α-MEM containing fetal bovine serum (10%), penicillin G sodium (100 μg/ml), streptomycin sulfate (100 μg/ml), and amphotericin B (0.25 μg/ml) (complete medium). CHO-K1 cells (50% confluent in 100-mm dishes) were transfected with 20 μg of plasmid DNA for wild type or mutated receptors using the Polybrene method as described previously(24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Transfected cultures were maintained in the presence of 800 μg G418/ml of complete medium for 10-14 days to select for plasmid containing cells, and individual clones were picked for propagation. Clonal lines were maintained under a selection pressure of 200 μg G418/ml and tested for the capacity to bind 125I-AII as described(24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Multiple clones expressing wild type or mutated receptor were obtained. Results for individual clones, chosen for full characterization and the internalization experiments described herein, were confirmed in one or two other clones.The authenticity of the data obtained from the clonal lines was also confirmed using transient transfections. For this, CHO-K1 cells at 80% confluence, in 35-mm dishes, were transfected using the lipofectamine method, as described by the manufacturer (Life Sciences). Cells were washed in OPTI-MEM, and for each well 1 μg of plasmid DNA was mixed with 8 μl of the lipofectamine reagent (2 mg/ml) in 200 μl of OPTI-MEM for 20 min. The mixture was diluted to 1 ml with OPTI-MEM and placed on the washed cells, which were returned to the incubator for 5 h. The DNA/lipofectamine solution was aspirated and replaced with 3 ml of complete media. Cells were cultured for 48 h and internalization assays performed. For each transfection, 125I-AII binding assays were performed in parallel on some wells to confirm transfection efficiency and receptor expression. The level of receptor expression, extrapolated by assuming similar dissociation constants established for the clonal cell lines, was approximately 400-600 fmol/mg protein for the wild type receptors and 100-400 fmol/mg protein for the mutants.AII Binding StudiesAII binding studies were performed on cultures of transfected CHO-K1 cells in 12-well culture plates as described previously(28Schorb W. Booz G.W. Dostal D.E. Conrad K.M. Chang K.C. Baker K.M. Circ. Res. 1993; 72: 1245-1254Crossref PubMed Scopus (364) Google Scholar). The AII receptor binding buffer contained 50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 4 mM KCl, 5 mM MgCl2, 1 mM CaCl2, 10 μg/ml Bacitracin, and 2 mg/ml D-glucose. Receptor affinity and density were determined by competition binding studies in the presence of 40 pM125I-AII and increasing concentrations (1 pM to 10 μM) of unlabeled AII.Determination of Receptor InternalizationClonal cell lines, stably expressing wild type or mutated receptors, were grown in triplicate to confluence in 12-well culture plates. The rate and degree of agonist-induced endocytosis was determined as follows: cells were washed three times with Hank's buffered salt solution, covered with 0.9 ml of AII receptor binding buffer (prewarmed to 37°C), and the plates returned to 37°C for 30 min. 125I-AII, in 100 μl of binding buffer, was added to a final concentration of 0.4-0.6 nM, and the incubation continued at 37°C for 2, 5, 10, and 20 min. At each time point, plates were chilled on ice and washed five times with 1.0 ml of ice-cold binding buffer to prevent further internalization and to remove unbound 125I-AII. Bound 125I-AII associated with non-internalized receptors in the plasma membrane was removed by two 40-s washes with 5 mM ice-cold acetic acid in 150 mM NaCl, pH 2.5. These acid washes were retained, and the internalized radioactivity was collected by adding 1.0 ml of 0.2 M NaOH, 0.25% sodium dodecyl sulfate to each well and washing with an additional 0.5 ml of the same solution. Radioactivity in the acid-sensitive and -insensitive fractions was measured with a LKB CompuGamma counter. After correction for background radioactivity associated with the parental untransfected CHO-K1 cells performed in parallel, an index of receptor internalization was obtained by expressing the acid-insensitive counts as a percentage of the total binding (acid-insensitive plus acid-sensitive) for each well. For transient transfections, the level of internalization was determined as above, but only at the 10-min time point.Analysis of the DataNon-linear regression analysis of the competition binding data was achieved using the computer software GraphPad Prism (GraphPad Software Inc.); dissociation constants (Kd) and receptor density (Bmax) were estimated as described(29Swillens S. Trends Pharmacol. Sci. 1992; 13: 430-434Abstract Full Text PDF PubMed Scopus (46) Google Scholar). For endocytosis assays, the percentage of internalized receptors was plotted against time, and the curves analyzed as one phase exponential associations using GraphPad Prism. The half-time (t in min) to reach a maximal level of internalization (Ymax in %) was determined for each association curve.Computer ModelingSecondary structure predictions for the carboxyl-terminal region of the AT1A receptor were obtained via the PHDsec server (Email: [email protected] ). Full description of the server and the prediction algothrim have been published(30Rost B. Sander C. J. Mol. Biol. 1993; 232: 584-599Crossref PubMed Scopus (2636) Google Scholar, 31Rost B. Sander C. Schneider R. Comput. Appl. Biosci. 1994; 10: 53-60PubMed Google Scholar, 32Rost B. Sander C. Proteins. 1994; 19: 55-72Crossref PubMed Scopus (1335) Google Scholar).RESULTScDNAs encoding wild type and mutated rat AT1A receptors were subcloned into the pRc/CMV mammalian expression vector and transfected into CHO-K1 cells. The various constructs are depicted in Fig. 1. Colonies resistant to neomycin (G418) were selected and propagated, and individual clonal lines expressing functional receptors were selected by the ability to bind 125I-AII. To determine if the expressed mutated receptors displayed affinities comparable to the wild type receptor, competition binding studies were performed. As summarized in Table 1 the wild type and assorted truncation, deletion, and point substitution mutants all bound 125I-AII with high affinity (Kd approximating 1 nM) indicating that these receptors attain a conformation necessary for high affinity recognition of AII. Dissociation constant values in the nanomolar range compare well with those previously reported for AT1 receptors expressed in cells and tissues(33Catt K.J. Robertson J.I. S Nicholls M.G. The Renin-Angiotensin System: Biochemistry and Physiology. Vol. 1. Gower Medical Publishing, London1993: 12.1-12.14Google Scholar). The level of receptor expression for the mutated receptors ranged between 145-840 fmol/mg protein (Table 1).Tabled 1 Open table in a new tab Using the appearance of acid-resistant radioactivity, after addition of 125I-AII at 37°C as an index of receptor internalization, we observed that wild type AT1A receptors expressed in CHO-K1 cells underwent rapid endocytosis from the plasma membrane (Fig. 2). Shown in Fig. 2 is the internalization profile for four separate clonal lines (T3, T5, T11, and T24) expressing the wild type receptor. Although the level of expression in these clones varies over a 10-fold range (T3, 3400 fmol/mg protein(26Thekkumkara T.J. Du J. Dostal D.E. Motel T.J. Thomas W.G. Baker K.M. Mol. Cell. Biochem. 1995; 146: 79-89Crossref PubMed Scopus (42) Google Scholar); T24, 667 fmol/mg protein(24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar); T11, 330 fmol/mg protein; T5, 310 fmol/mg protein), the kinetics of 125I-AII internalization, fitted as one phase exponential associations, were similar for all four clonal lines (T3, t 2.1 min, Ymax 80.9%; T5, t 2.3 min, Ymax 79.4%; T11, t 2.3 min, Ymax 80.6%; T24, t 2.5 min, Ymax 76.4%). This observation indicates that the level of receptor expression does not influence the degree of agonist-stimulated endocytosis and therefore internalization parameters for the mutant receptors can be directly compared despite varying levels of receptor expression (145-840 fmol/mg protein). One of the wild type receptor expressing clones, T24, was chosen for comparison with cells expressing mutated receptors. Also shown in Fig. 2 is the markedly reduced internalization (Ymax 16.2%) of TL314, confirming our previous observation (24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) that the carboxyl-terminal 45 amino acids of the AT1A receptor play a critical role in endocytosis. It is noteworthy that removal of 45 amino acids from the cytoplasmic tail did not prevent high affinity binding of AII, coupling to G-proteins, and appropriate activation of conventional signaling pathways(24Thomas W.G. Thekkumkara T.J. Motel T.J. Baker K.M. J. Biol. Chem. 1995; 270: 207-213Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).Figure 2:Endocytosis of the AT1A receptor is independent of receptor density but dependent upon an intact carboxyl terminus. CHO-K1 cells stably expressing varying densities of wild type AT1A receptor (T3, 3400 fmol/mg protein, filled circles; T5, 310 fmol/mg protein, open circles; T11, 330 fmol/mg protein, filled squares; T24, 667 fmol/mg protein, open squares) or a carboxyl-truncated AT1A receptor (TL314, crosses) were incubated with 125I-AII for 2-20 min at 37°C. At the indicated times, surface-bound and internalized 125I-AII were determined by acid washing as described under "Experimental Procedures." An index of internalization was calculated by expressing the internalized radioactivity (acid-resistant) as a percentage of the total binding (acid-resistant plus acid-susceptible). Data are means ± S.D. for three (TL314), six (T3, T5, and T11) or nine (T24) determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further define sites important for internalization (within this carboxyl-terminal region), we evaluated the capacity of another truncated receptor, TK333, to undergo AII-mediated endocytosis (Fig. 3). This mutant displayed a slower (t 6.1 min) and reduced amount (Ymax 46.3%) of internalization compared to the wild type T24 (t 2.5 min, Ymax 76.4%), implicating the terminal 26 amino acids in the internalization process. However, the level of internalization observed for TK333 was consistently greater than that observed for TL314 (Ymax 46.3 versus 16.2% (see Fig. 2)), which suggested that the region between Leu314 and Lys333 also contained an endocytotic determinant. To confirm this, a deletion mutant was constructed in which the terminal 30 amino acids(330-359) were fused to the first 314 residues, deleting amino acids 315-329. As shown in Fig. 3125I-AII was slowly (t = 8.6 min) internalized by this mutant, to a degree (Ymax = 40.9%) slightly less than that displayed by TK333. These observations suggest that there are two sites important for endocytosis within the carboxyl terminus of the AT1A receptor; one distal to Lys333 and another in the region 315-329.Figure 3:Two separate regions of the AT1A receptor carboxyl terminus are important for endocytosis. Internalization kinetics for the wild type AT1A receptor (filled circles) and truncated (TK333, open circles) and deletion (Del315-329, squares) mutants were obtained as described in the legend to Fig. 1. Data are the means ± S.D. from three separate experiments performed in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)While these observations were being made, Hunyady et al.(23Hunyady L. Bor M. Balla T. Catt K.J. J. Biol. Chem. 1994; 269: 31378-31382Abstract Full Text PDF PubMed Google Scholar) reported the inhibition of AT1A receptor internalization by mutation of amino acids within a carboxyl-terminal serine/threonine-rich region, in particu
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