Sequential Binding of Agonists to the β2 Adrenoceptor

Abstract

The β2 adrenoreceptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified β2AR. Here we show that upon catecholamine binding, β2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions. The β2 adrenoreceptor (β2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified β2AR. Here we show that upon catecholamine binding, β2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; AR, adrenoceptor; TMR-β2AR, β2AR labeled at Cys-265 with tetramethylrhodamine maleimide; TM, transmembrane. 1The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; AR, adrenoceptor; TMR-β2AR, β2AR labeled at Cys-265 with tetramethylrhodamine maleimide; TM, transmembrane. represent the largest family of membrane proteins in the human genome. They are responsible for the majority of cellular responses to hormones and neurotransmitters and are the largest group of targets for drug discovery. GPCRs are remarkably versatile biological sensors, responding to a broad spectrum of chemical entities ranging in size from a single photon of light, to ions, to small organic compounds, to peptides and protein hormones (1.Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Rhodopsin has long been used as a model system for studying the structure and mechanism of activation of GPCRs. It is the only GPCR for which a high resolution structure is available (2.Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4970) Google Scholar). Light-induced conformational changes have been elucidated by a series of elegant biophysical studies (3.Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1101) Google Scholar, 4.Dunham T.D. Farrens D.L. J. Biol. Chem. 1999; 274: 1683-1690Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 5.Altenbach C. Klein-Seetharaman J. Cai K. Khorana H.G. Hubbell W.L. Biochemistry. 2001; 40: 15493-15500Crossref PubMed Scopus (104) Google Scholar, 6.Altenbach C. Klein-Seetharaman J. Hwa J. Khorana H.G. Hubbell W.L. Biochemistry. 1999; 38: 7945-7949Crossref PubMed Scopus (93) Google Scholar, 7.Altenbach C. Cai K. Khorana H.G. Hubbell W.L. Biochemistry. 1999; 38: 7931-7937Crossref PubMed Scopus (114) Google Scholar, 8.Lin S.W. Sakmar T.P. Biochemistry. 1996; 35: 11149-11159Crossref PubMed Scopus (222) Google Scholar). Electron paramagnetic resonance spectroscopy studies provide evidence that photoactivation of rhodopsin involves a rotation and tilting of transmembrane domain 6 (TM6) relative to TM3 (3.Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1101) Google Scholar). Light-induced conformational changes have also been observed in the cytoplasmic domain spanning TM1 and TM2 (6.Altenbach C. Klein-Seetharaman J. Hwa J. Khorana H.G. Hubbell W.L. Biochemistry. 1999; 38: 7945-7949Crossref PubMed Scopus (93) Google Scholar) and the cytoplasmic end of TM7 and helix 8 (5.Altenbach C. Klein-Seetharaman J. Cai K. Khorana H.G. Hubbell W.L. Biochemistry. 2001; 40: 15493-15500Crossref PubMed Scopus (104) Google Scholar). Structural and biophysical studies on other GPCRs are more limited. Fluorescence spectroscopic analysis of β2ARs labeled with environmentally sensitive fluorescent probes detects movement of both TM3 and TM6 upon agonist binding (9.Gether U. Lin S. Ghanouni P. Ballesteros J.A. Weinstein H. Kobilka B.K. EMBO J. 1997; 16: 6737-6747Crossref PubMed Google Scholar). More detailed analysis of conformational changes in TM6 of the β2AR provide evidence for a rigid body motion similar to that observed upon activation of rhodopsin (10.Ghanouni P. Steenhuis J.J. Farrens D.L. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5997-6002Crossref PubMed Scopus (302) Google Scholar, 11.Jensen A.D. Guarnieri F. Rasmussen S.G. Asmar F. Ballesteros J.A. Gether U. J. Biol. Chem. 2001; 276: 9279-9290Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Additional support for movement of TM3 and TM6 in the β2AR comes from zinc cross-linking studies (12.Sheikh S.P. Vilardarga J.P. Baranski T.J. Lichtarge O. Iiri T. Meng E.C. Nissenson R.A. Bourne H.R. J. Biol. Chem. 1999; 274: 17033-17041Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and chemical reactivity measurements in constitutively active β2AR mutants (13.Javitch J.A. Fu D. Liapakis G. Chen J. J. Biol. Chem. 1997; 272: 18546-18549Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 14.Rasmussen S.G. Jensen A.D. Liapakis G. Ghanouni P. Javitch J.A. Gether U. Mol. Pharmacol. 1999; 56: 175-184Crossref PubMed Scopus (191) Google Scholar). Cysteine cross-linking studies in the M3 receptor provide evidence for the movement of the cytoplasmic ends of TM5 and TM6 toward each other upon agonist activation (15.Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Based on this limited set of experiments, it appears that the agonist-induced conformational changes leading to G protein activation for monoamine receptors are similar to those observed for rhodopsin. However, the process linking agonist binding to these conformational changes for rhodopsin may not be generalizable to the larger family of GPCRs for hormones and neurotransmitters because of the unique covalent interaction between rhodopsin and its agonist trans-retinal. Thus, the dynamic processes of agonist association and dissociation common to other GPCRs are not part of the activation process of rhodopsin, and the mechanism by which ligand binding leads to structural changes in GPCRs is poorly understood. A number of kinetic models have been developed to describe the process of agonist activation. These models are based on indirect measures of receptor conformation such as ligand binding affinity and G protein activation studies. Perhaps the most widely cited model is the extended ternary complex model (16.Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 17.Weiss J.M. Morgan P.H. Lutz M.W. Kenakin T.P. J. Theor. Biol. 1996; 181: 381-397Crossref PubMed Scopus (156) Google Scholar, 18.Kenakin T. FASEB J. 2001; 15: 598-611Crossref PubMed Scopus (352) Google Scholar). A simplified version of this model (referred to as the two-state model) is commonly used as a conceptual framework to discuss experimental results. The two-state model proposes that a receptor exists primarily in two states, the inactive state (R) and the active state (R*). In the absence of ligands, the level of basal receptor activity is determined by the equilibrium between R and R*. The efficacy of ligands reflects their ability to alter the equilibrium between these two states. Full agonists stabilize R*, whereas inverse agonists stabilize R. Although often discussed in the context of two receptor states, the extended ternary complex model is compatible with multiple receptor states, and several lines of experimental evidence support the existence of multiple states (18.Kenakin T. FASEB J. 2001; 15: 598-611Crossref PubMed Scopus (352) Google Scholar, 19.Kenakin T. Trends Pharmacol. Sci. 2003; 24: 346-354Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). We have used fluorescence lifetime spectroscopy to characterize the diversity of conformational states of the β2AR (20.Ghanouni P. Gryczynski Z. Steenhuis J.J. Lee T.W. Farrens D.L. Lakowicz J.R. Kobilka B.K. J. Biol. Chem. 2001; 276: 24433-24436Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). We found that the conformational states induced by full and partial agonists are distinguishable. Moreover, we found that a single catecholamine agonist induces or stabilizes at least two conformational states that are distinguishable from the unliganded state. Based on these studies, we proposed the existence of an intermediate state between the inactive (unliganded state) and the fully activated state. We now show that it is possible to observe and characterize an intermediate state by kinetic analysis. We use fluorescence spectroscopy to monitor agonist-induced conformational changes in purified β2AR over time. Our results support a mechanistic model for GPCR activation, where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions. Buffers—The buffers used are as follows: Buffer A, 100 mm NaCl, 20 mm HEPES pH 7.5, 0.1% dodecylmaltoside (Anatrace); Buffer B, Buffer A and 1 mm EDTA; Buffer C, Buffer A with 300 μm alprenolol (Sigma) and 1 mm CaCl2; Buffer D, Buffer A with 1 mm CaCl2; Buffer E, Buffer A with 0.01% cholesterolhemisuccinate. Receptor Purification and Labeling—β2AR was expressed in Sf9 cells and solubilized using methods described previously (21.Kobilka B.K. Anal. Biochem. 1995; 231: 269-271Crossref PubMed Scopus (145) Google Scholar). CaCl2 was added to a final concentration of 1 mm, and the detergent-solubilized β2AR was purified by M1-FLAG affinity chromatography (Sigma). The receptor was eluted from the M1-FLAG resin in Buffer B. The concentration of functional, purified receptor was determined using a saturating concentration (10 nm) of [3H]dihydroalprenolol as described previously (21.Kobilka B.K. Anal. Biochem. 1995; 231: 269-271Crossref PubMed Scopus (145) Google Scholar). FLAG-purified receptor was diluted to a concentration of 1 μm and labeled with tetramethylrhodamine-5-meleimide (Molecular Probes) at a final concentration of 1 μm for 1 h on ice. Labeled receptor was then purified by alprenolol-Sepharose chromatography as described previously (21.Kobilka B.K. Anal. Biochem. 1995; 231: 269-271Crossref PubMed Scopus (145) Google Scholar). The receptor was eluted from alprenolol-Sepharose with Buffer C and loaded directly onto M1-FLAG resin. The M1-FLAG resin was washed with Buffer D to remove free alprenolol and eluted with Buffer B. Two liters of Sf9 cells typically yield 500 μl of a 5 μm solution of tetramethylrhodamine-labeled β2AR (TMR-β2AR). Fluorescence Spectroscopy—Experiments were performed on a SPEX FluoroMax-3 spectrofluorometer with photon counting mode using an excitation and emission bandpass of 3.2 nm. Approximately 25 pmol of TMR-β2AR was desalted into 500 μl of Buffer E immediately before spectroscopy. For time course experiments, excitation was at 541 nm, and emission was monitored at 571 nm. Unless otherwise indicated, all experiments were performed at 30 °C, and the sample underwent constant stirring. Fluorescence intensity was corrected for dilution by ligands in all experiments and normalized to the initial value. All of the compounds tested had an absorbance of less than 0.01 at 541 and 571 nm in the concentrations used, excluding any inner filter effect in the fluorescence experiments. Spectra were corrected for fluorescence of the ligand when it was greater than 1% of the basal fluorescence of TMR-β2AR (only required for tyramine). cAMP Accumulation—The production of cAMP was determined by adenylyl cyclase activation FlashPlate assay (PerkinElmer Life Sciences) as described previously (22.Swaminath G. Lee T.W. Kobilka B. J. Biol. Chem. 2003; 278: 352-356Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Agonist-induced Internalization—HEK293 cells expressing FLAG-β2AR were cultured in poly-lysine-coated 12-well plates for 24 h. The cells were stimulated with different drugs for 10 min before fixing without permeabilization with 4% paraformaldehyde in phosphate-buffered saline (with Ca2+ and Mg2+). After blocking with 2.5% goat serum in phosphate-buffered saline (with Ca2+ and Mg2+), the cells were stained with Alexa-488 (Molecular Probes)-conjugated M1 antibody (Sigma) at a concentration of 1 μg/ml for 30 min. The unbound antibody was removed by washing four times with phosphate-buffered saline (with Ca2+ and Mg2+). The cells were harvested with 1% SDS in phosphate-buffered saline, and the intensity of Alexa-488 emission was measured on a FluorMax-3 spectrofluorometer. The excitation was at 485 nm, and the emission was from 495 to 580 nm with an integration time of 0.3 s/nm. The fluorescence intensity was normalized after subtracting the background from cells without M1 antibody. Statistical Analysis—Curve fitting and statistical analysis were performed using Prism (GraphPad Software, Inc.). Monitoring Agonist-induced Conformational Changes in the β2 AR with a Fluorescent Probe at the Cytoplasmic End of TM6—To study agonist-induced conformational changes in the β2AR, we monitor fluorescence intensity of purified β2AR labeled at Cys-265 with tetramethylrhodamine maleimide (TMR-β2AR) as a function of time. We have shown previously that it is possible to monitor agonist-induced conformational changes in β2ARs labeled at Cys-265 with either fluorescein maleimide (10.Ghanouni P. Steenhuis J.J. Farrens D.L. Kobilka B.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5997-6002Crossref PubMed Scopus (302) Google Scholar, 20.Ghanouni P. Gryczynski Z. Steenhuis J.J. Lee T.W. Farrens D.L. Lakowicz J.R. Kobilka B.K. J. Biol. Chem. 2001; 276: 24433-24436Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) or tetramethylrhodamine maleimide (23.Neumann L. Wohland T. Whelan R.J. Zare R.N. Kobilka B.K. Chembiochem. 2002; 3: 993-998Crossref PubMed Scopus (51) Google Scholar). An environmentally sensitive fluorophore covalently bound to Cys-265 is well positioned to agonist-induced conformational changes to G protein activation. Based on with rhodopsin (2.Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4970) Google Cys-265 is in the at the cytoplasmic end of the transmembrane 6 (TM6) studies have shown this of to be for G protein M. R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Moreover, with TM3 and that the agonist binding The of interaction between and the β2AR have been C. I. R. FASEB J. 3: PubMed Scopus Google Scholar, K. C. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, G. Ballesteros J.A. S. Chen Javitch J.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and are in The with in TM3 C. I. R. FASEB J. 3: PubMed Scopus Google and the catechol with in TM5 C. I. R. FASEB J. 3: PubMed Scopus Google Scholar, K. C. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, G. Ballesteros J.A. S. Chen Javitch J.A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). with the and the have both been to TM6 K. C. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). Conformational Changes in β2AR the of TMR-β2AR to a saturating concentration of two catecholamine norepinephrine and The time intensity were with and association functions. A was than a for the to norepinephrine In contrast, was between and for the to In the for norepinephrine is into its rapid and slow 6 the rapid of the for norepinephrine is similar to the observed for 0.3 of the rapid and slow of the conformational to an number of number of experiments. in a The of the in Conformational in the of TMR-β2AR to and norepinephrine be to the in norepinephrine but in Thus, conformational changes receptor interactions with the catechol the catechol and the in both and are whereas conformational changes with interactions between the receptor and the in are times is that the of binding determined by equilibrium binding The slow of of this interaction between the receptor and the that the conformational required to this interaction involves a the of and of norepinephrine on TMR-β2AR the of the in the slow of the conformational to shown in a rapid of is observed for both the slow for is a relative in Thus, it appears that the of norepinephrine to the β2AR but stabilizes a different conformational state. It is that the of the slow for norepinephrine is with that for However, we not the of norepinephrine to the in the β2AR as the of characterize the structural responsible for the rapid and slow conformational changes, we the to a panel of ligands that are related to and both have and both induce a in the intensity of TMR-β2AR The of the slow with the of an on the of the Moreover, the of the slow for which an is 6 than that of the slow for 20 and 10 Thus, receptor interactions with the conformational changes interactions between the and the β2AR are The for the of the Conformational the structural of the rapid of TMR-β2AR to we responses to and which from in the on the catechol not induce a in the fluorescence of Thus, the catechol structure is for the rapid conformational In catechol induces a rapid in TMR-β2AR, whereas is observed with The in the of the fluorescence to and catechol be to the interaction of the of and in TM3 of the β2AR this interaction on a similar rapid time 5 fluorescence experiments provide evidence for a sequential binding model in which the process of binding of a small organic agonist by kinetically distinguishable intermediate conformational states. model proposes that the unliganded receptor exists in a dynamic and state R that undergo transitions to an number of states. The of the unliganded state is based on fluorescence lifetime studies (20.Ghanouni P. Gryczynski Z. Steenhuis J.J. Lee T.W. Farrens D.L. Lakowicz J.R. Kobilka B.K. J. Biol. Chem. 2001; 276: 24433-24436Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) and the that R is more to U. Ballesteros J.A. R. Weinstein H. Kobilka B.K. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and B.K. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Moreover, R may undergo transitions to a state of activating the G the high basal activity observed for GPCRs C. S. S. P. Trends 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We that in the R the receptor not have a high affinity binding for the in the R the specific in agonist binding are not to the These interactions between the receptor and the agonist are such that each interaction the of the The process results in a series of intermediate states and and In our we that results from interactions between the catechol of the agonist and 5 and 6 of the receptor. when the with Our that binding of the catechol the binding of the is based on the that catechol induces a rapid conformational in TMR-β2AR In contrast, which an catechol but has the conformational that be by our on the of the for is greater than that for that the interaction between the and or stabilizes the interaction of the catechol with TM5 and The transitions from R to and are when the receptor the The from to is The and Conformational in the of the rapid and slow conformational responses that the different receptor states may have distinct Agonist binding interactions between the β2AR and activating signaling also interactions between the β2AR and and arrestins, which to G proteins for of the receptor L.M. R.J. J. Sci. 2002; PubMed Google Scholar). In an to the of the intermediate states proposed in the sequential binding model we the effect of the panel of compounds on G protein activation and interactions with and The state by binding of the catechol is not to activate the G However, the state by binding the catechol and the with cAMP in to is of that induced by Thus, is a partial agonist for the β2AR in interactions with In contrast, is only at β2AR that the is less efficient at interactions between the β2AR and epinephrine, and which stabilize both and are more in β2AR internalization. Thus, the slow from to appears to be required for interactions between the receptor and conformational changes in the β2AR induced by a panel of related ligands, we into the of linking ligand binding to receptor activation. Moreover, our studies that a single small organic ligand such as norepinephrine induce at least two kinetically and distinct conformational a rapid state of activating Gs and a slow state required for efficient agonist-induced internalization. interactions between the β2AR and are required for it is that the slow conformational state interactions between the β2AR and or both of these It is to that the of ligand binding may be responsible for the of the of receptor activation and Our results are in with studies on the receptor that different ligands induce distinct conformational states that in G protein activation and receptor L. M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). More binding studies on the receptor that an agonist with The rapid binding was with a cellular whereas the slow was required for cAMP signaling T. B. S. B. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Thus, it is that our that a single agonist induce or stabilize multiple distinct conformational states be generalizable to other those activated by peptides and protein hormones where are a larger number of of interaction between receptor and A of this conformational the of more and