The study identifies the alpha-1 subunit interaction domain (AID) as the major anchoring site for the beta subunit in voltage-dependent calcium channels.
β Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of α1-β subunit interaction have been localized recently to cytoplasmic domains of both subunits. The α1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all α1 subunits which is essential for the binding of β subunits. In order to further study the interaction of β subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the α1A subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized 35S-labeled β2 and β3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to β subunits. The affinity bead assay was also used to identify and characterize native β subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind β subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of β1a in skeletal muscle, β2 and β3 in cardiac muscle, and β1b, β3, and β4 in brain. The assays also demonstrate the AIDA beads bind to β subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of β subunits which is not always associated with α1 subunits. Also, β subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native β subunits from detergent and non-detergent tissue extracts illustrating that this domain on the α1 subunit is the major anchoring site for the β subunit. β Subunits of voltage-dependent Ca2+ channels play an important role in regulating Ca2+ channel function. The sites of α1-β subunit interaction have been localized recently to cytoplasmic domains of both subunits. The α1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all α1 subunits which is essential for the binding of β subunits. In order to further study the interaction of β subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the α1A subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized 35S-labeled β2 and β3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to β subunits. The affinity bead assay was also used to identify and characterize native β subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind β subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of β1a in skeletal muscle, β2 and β3 in cardiac muscle, and β1b, β3, and β4 in brain. The assays also demonstrate the AIDA beads bind to β subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of β subunits which is not always associated with α1 subunits. Also, β subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native β subunits from detergent and non-detergent tissue extracts illustrating that this domain on the α1 subunit is the major anchoring site for the β subunit. Voltage-dependent calcium channels play a critical role in the regulation of many cellular processes. A number of classes (T-, L-, N-, and P-type) of voltage-sensitive Ca2+ channels have been identified and distinguished on the bases of their electrophysiological and pharmacological properties(1Fox A.P. Nowycky M.C. Tsien R.W. J. Physiol. (Lond.). 1987; 394: 149-172Google Scholar, 2Fox A.P. Nowycky M.C. Tsien R.W. J. Physiol. (Lond.). 1987; 394: 173-200Google Scholar, 3Miller R. J. Biol. Chem. 1992; 267: 1403-1406Google Scholar). The L-type voltage-sensitive Ca2+ channel is involved in excitation-contraction coupling in skeletal and cardiac muscle while the T-type voltage-sensitive Ca2+ channel has been shown to be involved in pacemaker activity. The N-type (ω-conotoxin GVIA receptor) and P-type voltage-sensitive Ca2+ channels are found in both central and peripheral neurons and play an essential role in controlling neurotransmitter release from these neurons(4Smith S.J. Augustine G.J. Trends Neurosci. 1988; 11: 458-464Google Scholar). In addition to the regulation of transmitter release, the N-type Ca2+ channel also helps direct the migration of immature neurons(5Komuro H. Rakic P. Science. 1992; 257: 806-809Google Scholar). Both the skeletal muscle L-type and the neuronal N-type Ca2+ channels have been purified, and each of these Ca2+ channels contain common subunits (α1, α2δ, and β) and one variable subunit (γ and 95-kDa) (6Leung A.T. Imagawa T. Campbell K.P. J. Biol. Chem. 1987; 262: 7943-7946Google Scholar, 7Witcher D.R. De Waard M. Sakamoto J. Franzini-Armstrong C. Pragnell M. Kahl S.D. Campbell K.P. Science. 1993; 261: 486-489Google Scholar). The β1a and β3 subunits have been shown to associate with the purified dihydropyridine receptor and ω-conotoxin GVIA receptor, respectively. Biochemical studies have also demonstrated that the β subunit is tightly associated with the α1 subunit in Ca2+ channel complexes. Sequence analysis of β subunits have illustrated a lack of transmembrane segments in these proteins which suggest that these subunits are entirely cytoplasmic. β Subunits are also substrates for numerous protein kinases (8Jahn H. Nastainczyk W. Rohrkasten A Schneider T. Hofmann F. Eur. J. Biochem. 1988; 178: 535-542Google Scholar) and are thus regulated by intracellular signaling. Voltage-dependent Ca2+ channel subunits have now been divided into several classes based on their molecular properties. The pore-forming subunit, α1, has been separated into six classes, S, A, B, C, D, and E, while the β subunit has been separated into four classes; β1, β2, β3, and β4(9Birnbaumer L. Campbell K.P. Catteral W.A. Harpold M.M. Hofmann F. Horne W.A. Mori Y. Schwartz A. Snutch T.P. Tanabe T. Tsien R.W. Neuron. 1994; 13: 503-506Google Scholar). Also, other alternatively spliced products from these genes have been identified(10Perez-Reyes E. Wei X. Castellano A. Birnbaumer L. J. Biol. Chem. 1990; 265: 20430-20436Google Scholar, 11Powers P.A. Liu S. Hogan K. Gregg R.G. J. Biol. Chem. 1992; 267: 22967-22972Google Scholar, 12Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Google Scholar). So far, only one single gene seems to encode the α2δ subunit. Electrophysiological studies have demonstrated the importance of both the α2 and β subunits on the functional expression of α1 subunits (13Lacerda A.E. Kim H.S. Ruth P. Perez-Reyes E. Flockerzi V. Hofmann F. Birnbaumer L. Brown A.M. Nature. 1991; 352: 527-530Google Scholar, 14Singer D. Biel M. Lotan I. Flockerzi V. Hofmann F. Dascal N. Science. 1991; 253: 1553-1557Google Scholar, 15Varadi G. Lory P. Schultz D. Varadi M. Schwartz A. Nature. 1991; 352: 159-162Google Scholar, 16Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar, 17De Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Google Scholar). These subunits regulate the expression of the α1 subunit by inducing a conformational change in the structure of the α1 subunit and/or by targeting the Ca2+ channel to the membrane(18Neely A. Wei X. Olcese R. Birnbaumer L. Stefani E. Science. 1993; 262: 575-578Google Scholar, 19Nishimura S. Takeshima H. Hofmann F. Flockerzi V. Imoto K. FEBS Lett. 1993; 324: 283-286Google Scholar). Recently, it has been shown that the β subunit of voltage-dependent Ca2+ channels binds to a cytoplasmic sequence that is located between the first and second hydrophobic repeats of all α1 subunits(16Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar). This site has been named the alpha 1 subunit interaction domain or AID1 1The abbreviations used are: AIDα1 subunit interaction domainBIDβ subunit interaction domainGSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresisPBSphosphate-buffered salineCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acidMOPS4-morpholinepropanesulfonic acid. (20De Waard M. Witcher D.R. Pragnell M. Liu H.Y. Campbell K.P. Biophys. J. 1995; 68 (abstr.): A13Google Scholar). The AID site encompasses an 18-amino-acid sequence of which 9 amino acids are conserved. The corresponding interaction site on the β subunit has also been identified(17De Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Google Scholar). This site, a 30-amino-acid N-terminal region of the second conserved domain of the β subunit, has been named the beta subunit interaction domain or BID. The sequence of the BID is also very conserved between the different β subunit isoforms. It has been demonstrated by several point mutations of conserved amino acids in both epitopes that the AID and BID are required for the binding of β subunits and also the changes in activation kinetics, voltage dependence of activation and inactivation, and the increase in peak currents. Since both the AID and BID are necessary for the α1-β subunit interaction, an affinity bead assay has been developed to probe this subunit-subunit interaction(20De Waard M. Witcher D.R. Pragnell M. Liu H.Y. Campbell K.P. Biophys. J. 1995; 68 (abstr.): A13Google Scholar). The results reported here further characterize the molecular interaction between the α1 subunit and β subunit. We also demonstrate that native β subunits from various tissue extracts can be identified and purified with the use of AID affinity beads. α1 subunit interaction domain β subunit interaction domain glutathione S-transferase polyacrylamide gel electrophoresis phosphate-buffered saline 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid 4-morpholinepropanesulfonic acid. The TNT™-coupled reticulocyte lysate system was purchased from Promega. Isopropyl-1-thio-β-D-galactopyranoside is from Life Technologies, Inc., reduced glutathione from U. S. Biochemical Corp., 35Smethionine from Amersham, and glutathione-Sepharose and protein G-Sepharose from Pharmacia Biotech. Horseradish peroxidase-conjugated secondary antibodies were from Boehringer Mannheim. All other chemicals were of reagent grade. Triads from rabbit skeletal muscle were purified as described previously(21Sharp A.H. Imagawa T. Leung A.T. Campbell K.P. J. Biol. Chem. 1987; 262: 12309-12315Google Scholar). Proteins analyzed by SDS-PAGE (3-12% gradient gels) used the buffer system of Laemmli (22Laemmli U.K. Nature. 1970; Scholar). were with or to for analysis as described D.R. De Waard M. Campbell K.P. 1993; Scholar). The 35S-labeled β subunits were synthesized using the were number and β3 The of the β subunits were by acid by antibodies to β1b, β2, β3, and β4 were as described Kim H. H. Franzini-Armstrong C. R. Campbell K.P. Neuron. 1991; 7: Scholar). rabbit antibodies were a of the region of the β1a P. Rohrkasten A. Biel M. E. S. Flockerzi V. Hofmann F. Science. and antibodies were the of the M. Sakamoto J. S.D. Campbell K.P. FEBS Lett. 1991; Scholar). rabbit antibodies and were the of the β2 R. D. M. Biel M. Dascal N. Hofmann F. Flockerzi V. J. 1992; 11: Scholar, E. Castellano A. Kim P. E. A.E. Wei X. Birnbaumer L. J. Biol. Chem. 1992; 267: Scholar). antibodies were the of the β3 subunit R. D. M. Biel M. Dascal N. Hofmann F. Flockerzi V. J. 1992; 11: Scholar, A. Wei X. Birnbaumer L. Perez-Reyes E. J. Biol. Chem. 1993; and antibodies were the amino acid of the β4 A. Wei X. Birnbaumer L. Perez-Reyes E. J. Biol. Chem. 1993; Scholar). All of the rabbit antibodies were affinity-purified from of each to as described D.R. P. Kahl S.D. T. P. M. Campbell K.P. J. Biol. Chem. 1994; Scholar). Also, β1a and antibodies were affinity-purified from A.H. Campbell K.P. J. Biol. Chem. Scholar) using of the β1a and subunits of the skeletal muscle dihydropyridine receptor, respectively. affinity-purified antibodies were used in the of cardiac The of affinity-purified antibodies were to was on the GST fusion protein the AIDA using the system as described M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar). The were used to the GST fusion and The mutations were by sequence the 1988; Scholar) for control and AIDA fusion proteins and the α1A subunit motif that binds the β M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar) were into were and to D.R. De Waard M. Campbell K.P. 1993; Scholar). The were and in and for The was for in a The of the control GST fusion protein was with glutathione-Sepharose beads. The glutathione-Sepharose beads were with and the fusion proteins were with The was for the and AIDA GST fusion proteins of these fusion proteins were in these GST fusion the from the first were in of for the was by as described was to the to a of and with glutathione-Sepharose beads for the glutathione-Sepharose beads were with and the fusion proteins were with glutathione in AIDA GST fusion and AIDA GST fusion proteins were expressed and purified using glutathione-Sepharose as described the tissue homogenates used in the rabbit skeletal muscle, cardiac muscle, and were in a in a in the buffer 1 and several A and for The tissue homogenates were for and for in a The were and 1 to in a buffer with as described The were on for 1 and for in a to The solubilized homogenates were with the GST fusion proteins to beads the the beads were with and Proteins to the beads were analyzed by SDS-PAGE and were also by rabbit skeletal and with a buffer that and no These homogenates were also with AIDA or AIDA GST fusion proteins to beads and analyzed by SDS-PAGE and AIDA GST fusion and AIDA GST fusion protein were coupled to beads in for and a protein of was four with and the on the beads were with for The was in and of skeletal muscle triads were solubilized a protein of in a buffer 1 and for 1 The was for in a The was with and was a for The was with and and the skeletal muscle β subunit was from the with of buffer and The were with 1 the interaction between the α1 and β we have developed an in vitro binding Waard M. Witcher D.R. Pragnell M. Liu H.Y. Campbell K.P. Biophys. J. 1995; 68 (abstr.): A13Google Scholar). In this affinity the AIDA was expressed as a 50-amino-acid GST fusion protein and used as a for in vitro and native β subunits. characterize the interaction of the AIDA with different β we the of several AID GST fusion each point mutations tyrosine to bind to different β subunits 392 is one of several shown to be important for the α1-β subunit These GST fusion and along with GST were coupled to glutathione-Sepharose beads to form affinity beads. Both in vitro 35S-labeled β2 and β3 subunits were used as the for the affinity binding assay The results of the binding assay to the AIDA GST fusion the binding of the AIDA fusion proteins to both 35S-labeled β2 and β3 were to was with an assay with 35S-labeled M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google a of the of both 35S-labeled β2 and β3 subunits. with the that this tyrosine is critical for the α1-β interaction, the affinity beads a of the only and of the 35S-labeled β2 and β3 respectively. Also, affinity beads a of the and of the 35S-labeled β2 and β3 respectively. These results demonstrate that the mutations in tyrosine 392 a on the of both 35S-labeled β2 and β3 to bind to the AIDA affinity beads. the in the affinity between the β subunit and the affinity beads that the hydroxyl group in tyrosine 392 an important role in the molecular interaction between the α1 and β subunits in voltage-dependent Ca2+ In order to the of β subunits from solubilized skeletal muscle triads to bind the AID, this was expressed as a 50-amino-acid GST fusion The AIDA GST fusion protein was coupled to glutathione-Sepharose beads and used as the for the binding of native β subunits in solubilized skeletal muscle A GST fusion which contains a single amino acid change in the and GST were also and coupled to glutathione-Sepharose beads to form affinity beads. The interaction between the and the β subunit was shown to be reduced using an assay or in expression studies with M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar). We have demonstrated the of the AIDA bead assay with in vitro 35S-labeled β Waard M. Witcher D.R. Pragnell M. Liu H.Y. Campbell K.P. Biophys. J. 1995; 68 (abstr.): A13Google Scholar). that beads are of binding β subunits from solubilized skeletal muscle Immunoblot analysis with affinity-purified antibodies the subunit of the skeletal muscle dihydropyridine receptor from A.H. Campbell K.P. J. Biol. Chem. Scholar) that the α1 subunit was only in solubilized skeletal muscle triads and of the AIDA bead assay but not with the affinity beads These results suggest that the beads bind only to β subunits that are not associated with in native the role of AID as the β anchoring Immunoblot analysis with affinity-purified antibodies the β1a subunit from also that the beads were to bind the β1a subunit from the solubilized skeletal muscle The affinity-purified antibodies identified a a molecular of and a of the β1a subunit to the beads demonstrated that the of the was a form of the β1a subunit not The a molecular of was identified by other but not affinity-purified antibodies on suggesting that this protein is a of the It is also that this protein is an alternatively spliced form of or β subunit. This protein is not in the purified skeletal muscle dihydropyridine receptor which that this β subunit associate with a different α1 subunit in skeletal muscle or a peripheral in skeletal muscle Both GST and the beads not bind β subunits It has been shown that the AID site the β subunit to the α1 M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar, 17De Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Google Scholar). the existence of other sites of interaction between the β subunit and the α1 R. N. Schneider T. A. Wei X. Stefani E. Birnbaumer L. Neuron. 1994; 13: Scholar). Our results that native β subunits are associated with the AID no subunits can bind to the subunit This that the sites are α1 subunits bind with affinity to sites other the BID on β subunits. data the that the interaction site identified by Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar) and De Waard Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Google Scholar) are the only major affinity sites that the β subunit to the α1 subunit and that other interaction sites in β subunits are low affinity and bead assays were also on skeletal muscle SDS-PAGE of the proteins to the affinity beads that the glutathione-Sepharose beads of and fusion proteins The beads, but not the GST or beads, specifically β1a subunit from the skeletal muscle The β1a subunit was by analysis using affinity-purified antibodies from These antibodies were the region of the β1a subunit. The in the of the β1a subunit on the polyacrylamide gel and The β1a subunit in the and was by analysis the of the β1a subunit with the affinity beads. the affinity-purified antibodies identified a of a β subunit which the molecular of The subunit was only with the affinity beads and not in the skeletal muscle antibodies other β subunit β3, and not the presence of these on These results the results of analysis which have shown only the β1a in skeletal P. Rohrkasten A. Biel M. E. S. Flockerzi V. Hofmann F. Science. Scholar). the use of the affinity bead the of β subunits in detergent cardiac muscle which to the beads was that the glutathione-Sepharose beads of and fusion proteins analyzed on SDS-PAGE and with Immunoblot analysis of from polyacrylamide and with affinity-purified antibodies to other β subunit β3, and demonstrated that both β2 and β3 were in cardiac tissue and to the beads Immunoblot analysis not or β4 subunits using this affinity bead assay suggesting that these β subunits are not expressed in Our data are in with analysis that the expression of both β2 and β3 subunit in cardiac R. D. M. Biel M. Dascal N. Hofmann F. Flockerzi V. J. 1992; 11: Scholar, E. Castellano A. Kim P. E. A.E. Wei X. Birnbaumer L. J. Biol. Chem. 1992; 267: Scholar). These results demonstrate that both the β2 and β3 subunits are the protein in cardiac tissue and are with the that β2 be associated with the subunit in cardiac the β3 subunit is also associated with the or other α1 subunit in cardiac muscle to be the results of the AIDA affinity bead assay from The glutathione beads used in the assay of AIDA GST fusion and the GST fusion protein as demonstrated by the polyacrylamide gel that affinity-purified antibodies from and identified β3 and on analysis of the affinity beads. GST and beads not bind of β subunits from the Both and β2 not be by analysis using this both have been shown to be in M. Sakamoto J. S.D. Campbell K.P. FEBS Lett. 1991; Scholar, R. D. M. Biel M. Dascal N. Hofmann F. Flockerzi V. J. 1992; 11: Scholar, E. Castellano A. Kim P. E. A.E. Wei X. Birnbaumer L. J. Biol. Chem. 1992; 267: the of protein expression of these β subunits have these subunits to with this affinity bead Also, the between the analysis and the of protein by this be to and/or in β subunit In order to the protein expression of the four β subunit in different of the rabbit bead assays were on tissue extracts from the and The of tissue from each region was with the buffer the use of affinity-purified antibodies from analysis that was in both the and very in but or in the and The protein expression was in the in the for the subunit. The of protein expression of this β subunit the of the for this β subunit on M. Sakamoto J. S.D. Campbell K.P. FEBS Lett. 1991; Scholar). Immunoblot analysis of the bead from extracts not the subunit This that in extracts the subunit is the for the affinity bead Immunoblot analysis of the β subunits to the affinity beads from different also demonstrated that both β3 and β4 subunits were expressed in all The β3 subunit to be expressed a the to both and β4 subunits. affinity-purified antibodies β2 were also used on a no was in of the different This that the β2 subunit is or that are using this assay the rabbit brain. the existence of β subunits that were not into voltage-sensitive Ca2+ channel bead assay was with tissue homogenates which were extracted using a low salt buffer and no we have demonstrated in the the AIDA in the α1 subunit is of binding native β subunits from skeletal muscle, cardiac muscle, and brain. the buffer used to the different salt and the of in vitro 35S-labeled β subunits to the AIDA GST fusion protein be by these low salt and no were used in the tissue that the affinity beads to native β1a subunits that were in this skeletal muscle using analysis with affinity-purified antibodies from The beads also the β3 subunit from low salt extracts the of the bead both GST and beads not bind the β subunits in the tissue The β1a and β3 subunits were not in the homogenates and of each analyzed on of the bead These results that are β subunits which are not tightly with the α1 subunits. studies with the α1 and β subunits in have shown that the β subunit the of the α1 subunit by the and voltage dependence and of the Ca2+ A.E. Kim H.S. Ruth P. Perez-Reyes E. Flockerzi V. Hofmann F. Birnbaumer L. Brown A.M. Nature. 1991; 352: 527-530Google Scholar, 14Singer D. Biel M. Lotan I. Flockerzi V. Hofmann F. Dascal N. Science. 1991; 253: 1553-1557Google Scholar, 15Varadi G. Lory P. Schultz D. Varadi M. Schwartz A. Nature. 1991; 352: 159-162Google Scholar, 16Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Google Scholar, 17De Waard M. Pragnell M. Campbell K.P. Neuron. 1994; 13: 495-503Google Scholar). These studies have shown that β subunits which are tightly associated with α1 subunits play an important role in the of the pore-forming α1 subunit. β subunits also play a role in Ca2+ It is that β subunits be to the to bind with α1 subunits or of the or by the of intracellular as protein These β subunits associate with α1 subunits in the to increase their Ca2+ channel and their voltage dependence and AIDA beads were used to β subunits from solubilized skeletal muscle In SDS-PAGE demonstrated that a protein of a molecular of from the AIDA low in 9 and Immunoblot analysis with affinity-purified antibodies from the of the protein as the β1a subunit the use of the AIDA affinity the purified β1a subunit to bind to the affinity beads but not the beads not In results demonstrate that both and mutations in the tyrosine of the AID have a on the of the AID to bind to β subunits. of the the hydroxyl group the subunit-subunit a of only the hydroxyl group on the tyrosine a on β subunits binding to the suggesting that this hydroxyl group play an important role in the molecular between α1 and β subunits. Our results also that the AID binds to native β subunits in both detergent and non-detergent tissue This not only a of different β subunits from various but also that β subunits not always be tightly associated with α1 subunits. We C. D. and V. for their
Witcher et al. (Sat,) studied this question.