Agonist-regulated Interaction between α2-Adrenergic Receptors and Spinophilin

Abstract

Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical α2A-adrenergic receptor (α2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999)J. Biol. Chem. 274, 19894–19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether α2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the α2AAR (Val217-Ala377), α2BAR (Lys210-Trp354), and α2CAR (Arg248-Val363) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169–255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because α2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α2AR localization but also to agonist modulation of α2AR signaling. Previously, we demonstrated that the third intracellular (3i) loop of the heptahelical α2A-adrenergic receptor (α2AAR) is critical for retention at the basolateral surface of polarized Madin-Darby canine kidney II (MDCKII) cells following their direct targeting to this surface. Findings that the 3i loops of the D2 dopamine receptors interact with spinophilin (Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999)J. Biol. Chem. 274, 19894–19900) and that spinophilin is enriched beneath the basolateral surface of polarized MDCK cells prompted us to assess whether α2AR subtypes might also interact with spinophilin. [35S]Met-labeled 3i loops of the α2AAR (Val217-Ala377), α2BAR (Lys210-Trp354), and α2CAR (Arg248-Val363) subtypes interacted with glutathione S-transferase-spinophilin fusion proteins. These interactions could be refined to spinophilin amino acid residues 169–255, in a region between spinophilin's F-actin binding and phosphatase 1 regulatory domains. Furthermore, these interactions occur in intact cells in an agonist-regulated fashion, because α2AAR and spinophilin coimmunoprecipitation from cells is enhanced by prior treatment with agonist. These findings suggest that spinophilin may contribute not only to α2AR localization but also to agonist modulation of α2AR signaling. α2 adrenergic receptor glutathioneS-transferase Madin-Darby canine kidney polyacrylamide gel electrophoresis protein-phosphatase 1 spinophilin (neurabin II) third intracellular loop mitogen-activated protein polyvinylidene difluoride hemagglutinin Dulbecco's modified Eagle's medium phosphate-buffered saline phenylmethylsulfonyl fluoride reduced glutathione n-dodecyl-β-d-maltoside cholesteryl hemi-succinate cyclohexylamino-1-propane sulfonic acid The three α2-adrenergic receptor (α2AR)1subtypes are members of the type II, biogenic amine-binding, G protein-coupled receptor family. These receptor subtypes all couple via the Gi/Go family of GTP-binding proteins to the inhibition of adenylyl cyclase, inhibition of voltage-dependent calcium channels, potentiation of potassium currents via G protein-coupled, inwardly rectifying potassium channels, activation of phospholipase D, and activation of MAP kinase in native cells (1Kobilka B. Annu. Rev. Neurosci. 1992; 15: 87-114Crossref PubMed Scopus (315) Google Scholar, 2Limbird L.E. FASEB. J. 1988; 2: 2686-2695Crossref PubMed Scopus (277) Google Scholar, 3Jinsi A. Paradise J. Deth R.C. Eur. J. Pharmacol. 1996; 302: 183-190Crossref PubMed Scopus (30) Google Scholar, 4Richman J.G. Regan J.W. Am. J. Physiol. 1998; 43: C654-C662Crossref Google Scholar). In heterologous cell systems, these receptors also couple to the activation of a variety of signaling molecules, including Ras (5Alblas J. van Corven E.J. Hordijk P.L. Milligan G. Moolenaar W.H. J. Biol. Chem. 1993; 268: 22235-22238Abstract Full Text PDF PubMed Google Scholar, 6Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 7Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), p70S6 kinase (8Wilson M. Burt A.R. Milligan G. Anderson N.G. J. Biol. Chem. 1996; 271: 8537-8540Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), MAP kinase (9Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and phospholipase D (11MacNulty E.E. McClue S.J. Carr I.C. Jess T. Wakelam M.J. Milligan G. J. Biol. Chem. 1992; 267: 2149-2156Abstract Full Text PDF PubMed Google Scholar). Although all three α2ARs appear to activate similar signaling pathways, differences in the cellular trafficking of these subtypes have been reported, both in naive cells and following agonist activation. Subtype-selective differences in agonist-elicited α2AR redistribution have been noted in several experimental systems (12Von Zastrow M. Daunt D.A. Barsh G. Kobilka B.K. J. Biol. Chem. 1992; 268: 763-766Abstract Full Text PDF Google Scholar, 13Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1994; 269: 18448-18452Abstract Full Text PDF PubMed Google Scholar, 14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 15Kurose H. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10093-10099Abstract Full Text PDF PubMed Google Scholar, 16Eason M.G. Liggett S.B. J. Biol. Chem. 1992; 267: 25473-25479Abstract Full Text PDF PubMed Google Scholar, 17Jones S. Leone S. Bylund D.B. J. Pharmacol. Exp. Ther. 1990; 254: 294-300PubMed Google Scholar, 18Olli-Lahdesmaki T. Kallio J. Scheinin M. J. Neurosci. 1999; 19: 9281-9288Crossref PubMed Google Scholar). The α2BAR subtype is readily internalized following agonist activation, whereas the α2AAR subtype typically is not (14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 18Olli-Lahdesmaki T. Kallio J. Scheinin M. J. Neurosci. 1999; 19: 9281-9288Crossref PubMed Google Scholar). The α2CAR subtype has not been explored in as much detail with regard to agonist-elicited redistribution because of its considerable accumulation intracellularly (14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar). The α2AR subtypes also manifest different trafficking itineraries in polarized Madin-Darby canine kidney II (MDCKII) cells, even in the absence of agonist treatment. The α2AAR subtype is targeted directly to the basolateral surface (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar), whereas the α2BAR subtype is delivered randomly to both the apical and basolateral surfaces but is selectively retained on the basolateral surface (t12 = 10–12 h) in contrast to its rapid loss from the apical surface (t12 = 5–15 min) (20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These findings suggest that there is a molecular mechanism responsible for the selective retention of the α2BAR on the basolateral sub-domain of MDCK cells, probably a retention mechanism shared by the basolaterally targeted α2A- and α2CAR subtypes (20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Although α2CARs, like α2AARs, are directly targeted to and retained on the basolateral subdomain, a significant proportion of these receptors is identifiable in an intracellular pool at steady state (14Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar, 18Olli-Lahdesmaki T. Kallio J. Scheinin M. J. Neurosci. 1999; 19: 9281-9288Crossref PubMed Google Scholar, 20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar); the functional relevance of this intracellular α2CAR pool has yet to be clarified. Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the α2AR subtypes. For example, deletion of this loop in the α2AAR subtype (Δ3i α2AAR) results in accelerated basolateral receptor turnover (t12 ≅ 4.5 h) when compared with that for the wild-type receptor or with α2AAR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t12 of 10–12 h) (21Keefer J.R. Kennedy M.E. Limbird L.E. J. Biol. Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar). Similarly, the Δ3i α2BAR is not enriched at the basolateral surface of MDCKII cells at steady state (22Saunders C. Limbird L.E. Mol. Pharmacol. 2000; 57: 44-52PubMed Google Scholar). Based on our findings that the α2BAR is rapidly removed from the apical surface following random delivery and that removal of the 3i loops of the α2A- and α2βAR subtypes accelerates surface turnover of these receptors, we hypothesize that α2ARs interact, via their 3i loops, with protein(s) enriched beneath the basolateral surface of MDCKII cells to stabilize their steady-state localization. Consequently, we were particularly intrigued by recent findings that the 3i loop of another Gi/Go-coupled G protein-coupled receptor, the D2 dopamine receptor, interacts with spinophilin (23Smith F.D. Oxford G.S. Milgram S.L. J. Biol. Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 25Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (391) Google Scholar), and that this protein is enriched beneath the basolateral surface of polarized MDCK cells (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In addition, the multiple protein-interacting domains within spinophilin (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) suggest that its interaction with the receptor may facilitate the formation of a signaling complex to modulate signaling or recruitment of other proteins to a functional microdomain. The present studies were undertaken to identify whether spinophilin interacts with the 3i loops of the α2AR subtypes and, if so, if these interactions are regulated by agents that modify α2AR function. The pGEMEX-2 vector and TnT in vitro translation kit were from Promega (Madison, WI). The [35S]methionine (1000 Ci/mmol, at 10 mCi/ml) was purchased from PerkinElmer Life Sciences (Boston, MA). PVDF nylon membranes were from Millipore (Bedford, MA). The fast protein liquid chromatography and DEAE-Sephacel columns were from Amersham Pharmacia Biotech (Piscataway, NJ). Dodecyl-β-maltoside and cholesteryl-hemisuccinate were purchased from Calbiochem (San Diego, CA) and Sigma Chemical Co. (St. Louis, MO), respectively. Antibodies against the HA epitope engineered into the α2AR structures was obtained from BABCo (mouse) or from Roche Molecular Biochemicals (rat and mouse). Mouse anti-Myc antibodies were purchased from CLONTECH (Palo Alto, CA). Protein A-agarose was from Vector (Burlingame, CA). were purchased from MA). and antibodies were from Amersham Pharmacia for was Amersham Pharmacia and antibodies were from Molecular MDCKII cells were at and on in Dulbecco's modified Eagle's medium with and and 10 at (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar) with for these cells a and with apical and basolateral surfaces by that have and that the apical and basolateral are from another the (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). For these of is to the apical and for 1 at by of the medium in of the apical and basolateral from and we from of antibodies were by of glutathioneS-transferase fusion proteins to spinophilin amino as by M. S. B.E. R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Antibodies were from by were by of and 1 of in in a 10 of a chromatography fusion protein in of was the and with for at The was with of as on the were by with 1 for 1 at with The was with and at was in the was to the was to the and with for at The was three with with in and with was with of and into of 1 to the was and into an with a against the of the fusion was with the as by for at The from this was and an as and as the was to be by protein of in and were via and MDCKII cells the α2AR subtypes were on as and with saline with 1 and and for with at or with at by with with in was whereas the α2AR localization was following and For we for of the polarized MDCKII cells were in in to the cell surface of the for and in and in for 1 was to the cell of and for 1 at or h) at Mouse antibodies were at and antibodies were at a of MDCKII cells were three for in at The antibodies were or or and were with the cells for 1 at were three for in and a with and a were on a in the were a at The residues to the 3i loops of the α2AAR D.A. Limbird L.E. J. Biol. Chem. 1990; Full Text PDF PubMed Google the α2BAR Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google and the α2CAR S. J. Biol. Chem. Full Text PDF PubMed Google were into the vector within the of the the protein 10 were in were via into the region of the α2AAR 3i loop and into the were by The loop fusion proteins and loops were and [35S]Met-labeled and as of TnT was to 1 of amino acid of 1 of TnT of [35S]methionine (1000 Ci/mmol, at 10 and 1 of 1 of the was and the was to with The was for at were and by gel electrophoresis and The was of the gel and in were that an of [35S]Met-labeled 3i loop as fusion proteins were with spinophilin amino acid and and in were at to an of or fusion protein was with the of 1 and to for at were by at and in 1 1 1 10 by by for three on of a in was to the of a and for 1 at with was to a and with of of in and with of or fusion protein was from the by of 10 acid in protein was and into an of fusion protein were with to [35S]Met-labeled or 3i loop with was to this for at and the by The was to with was by of the 3i loop into and of the by The of interaction was by and the to 3i loop via in cells were at cells on and in with and and 10 at The following cells were Molecular to the with an of of The and and were in and at their the with the to the hemagglutinin as (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). spinophilin was in and with a the was the cells were in and with or not for at of signaling by α2ARs inhibition of adenylyl or of MAP typically is following with for (9Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). were in and into 1 1 10 and were by at at for and hemi-succinate 1 1 10 and by a by in a The of a for 1 at was as the of this was by a with of protein A-agarose with α2AAR or spinophilin was following the of or anti-Myc at a and at for 1 The complex was by following a to protein proteins were removed by the protein three in 1 1 10 and and at were with the of and to for were via to an with a of 1 for in sulfonic acid and to PVDF membranes were for in saline with and The was at a of or anti-Myc in and at for were three for with and to or as at a in for at were three for in with Pharmacia for and to for from to is a protein P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (391) Google Scholar) of an F-actin binding a region M. S. B.E. R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Allen P.B. T. Greengard P. 1999; PubMed Scopus Google Scholar, L. Feng J. K. Allen P.B. Greengard P. Neurosci. 1999; 2: PubMed Scopus Google a binding and a terminus that a of domains in Satoh (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) that spinophilin was to the lateral sub-domain in polarized MDCK the α2A-adrenergic receptor also is enriched on the lateral sub-domain of these cells (19Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 20Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and is a against the that the HA epitope in the α2AAR was against amino in spinophilin M. S. B.E. R.J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), a region that has to spinophilin's in the against via considerable of spinophilin at the lateral surface of these polarized cells, of Satoh (24Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). The of of the α2AAR and spinophilin in the lateral of MDCKII cells is demonstrated by the considerable of present in the results were of the α2BAR subtype and spinophilin not be that spinophilin also is including in a of with spinophilin in polarized MDCKII cells were and polarized on for The of the receptor and spinophilin was as antibodies were and The of in the of the were a = via a at in both the and to the as in the to the of the (23Smith F.D. Oxford G.S. Milgram S.L. J. Biol. Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) have via and gel that the 3i loops of the D2 dopamine receptor and interact with spinophilin in the region between the F-actin binding and domains Consequently, we fusion proteins of spinophilin between amino and in that the 3i loop of of the α2AR subtypes is to interact with α2AR 3i loops interacted with but not with the of the 3i loop of the α2AAR was with a fusion protein of amino region was because the region of with the a protein H. Obaishi H. Satoh A. Wada M. K. Satoh K. Nishioka H. Y. Mizoguchi A. Takai Y. J. Biol. 1997; PubMed Scopus Google because there is a of amino between these protein family members within this was of considerable to the of the region of to interact with the α2AAR 3i in or interaction was between the α2AAR 3i loop and when compared with Furthermore, binding to the 3i loop is demonstrated by the region of in binding of the loop to this region is readily to this to was of to whether or not these in vitro via fusion protein could be in the of a For these we α2AAR and spinophilin in the of cells were with or prior to of the cell membranes and with a that receptor in a functional L. J.G. Limbird L.E. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). in of spinophilin to the of the in cells both receptor and spinophilin 1 is an from cells only the α2AAR also has been from cells with only that not the of to an interaction with not is the of the α2AAR to the of α2AAR in with that regulated interactions with spinophilin could contribute both to receptor localization and of by studies have demonstrated an interaction between the third intracellular loops of the α2AR subtypes and spinophilin and findings of in that the 3i loops of the D2 dopamine receptor and interact with spinophilin fusion proteins (23Smith F.D. Oxford G.S. Milgram S.L. J. Biol. Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). 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Chem. 1994; 269: 16425-16432Abstract Full Text PDF PubMed Google Scholar) and (22Saunders C. Limbird L.E. Mol. Pharmacol. 2000; 57: 44-52PubMed Google Scholar). are to M. for critical and to for also are for the of and with and Milgram, of and in of these the shared of the other members of the Limbird