Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons

The sodium channel from rat brain is a complex of alpha (260 kd), beta 1 (36 kd), and beta 2 (33 kd) subunits. The alpha and beta 2 subunits are linked by disulfide bonds. The earliest biosynthetic precursor of the alpha subunit is a 203 kd core polypeptide with sufficient high-mannose carbohydrate chains to increase its apparent size to 224 kd. It is processed to 224 kd and 249 kd precursor forms containing complex carbohydrate chains before it achieves the mature size of 260 kd. Most newly synthesized alpha subunits are not disulfide-linked to beta 2 subunits, but remain as a metabolically stable pool of intracellular subunits. alpha subunits disulfide-linked to beta 2 are found preferentially at the cell surface. A possible role for this intracellular pool as a rate-limiting step in the regulation of the cell surface density and localization of sodium channels in developing neurons is proposed.     

Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.     

The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits

The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations.     

Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

Palmitylation, sulfation, and glycosylation of the alpha subunit of the sodium channel. Role of post-translational modifications in channel assembly

Antibodies to the alpha and beta 2 subunits and site-directed antibodies that distinguish alpha subunits of the RI and RII subtypes have been used to study the biosynthesis and assembly of sodium channels. The RII sodium channel subtype is preferentially expressed in rat brain neurons in primary cell culture. Post-translational processing of alpha subunits includes incorporation of palmityl residues in thioester linkage and sulfate residues attached to oligosaccharides. The incorporation of [3H] palmitate into alpha subunits is inhibited by tunicamycin, indicating that it occurs in the early stages of biosynthesis but after co-translational glycosylation. Mature alpha subunits are attached to beta 2 subunits through disulfide bonds within 1 h after synthesis and up to 30% can be specifically immunoprecipitated from the cell surface with antibodies against the beta 2 subunits by 4 h after synthesis. The remaining alpha subunits remain in an intracellular pool. The alpha subunits synthesized in the presence of castanospermine and swainsonine have reduced apparent size. Castanospermine prevents incorporation of approximately 81% of the sialic acid of the alpha subunit and inhibits sulfation but not palmitylation. Although inhibition of glycosylation with tunicamycin blocks assembly of functional sodium channels, castanospermine and swainsonine do not prevent the covalent assembly of alpha and beta 2 subunits or the transport of alpha beta 2 complexes to the cell surface, and sodium channels synthesized under these conditions have normal affinity for saxitoxin. Thus, the extensive processing and terminal sialylation of oligosaccharide chains during maturation of the alpha subunit is not essential. A kinetic model for biosynthesis, processing, and assembly of sodium channel subunits is presented.     

Hydrophobic properties of the beta 1 and beta 2 subunits of the rat brain sodium channel

Voltage-sensitive sodium channels purified from rat brain in functional form consist of a stoichiometric complex of three glycoprotein subunits, alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. The hydrophobic properties of these three subunits were examined by covalent labeling with the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which labels transmembrane segments in integral membrane proteins. All three subunits of the sodium channel were labeled by [125I]TID when the purified protein was solubilized in mixed micelles of Triton X-100 and phosphatidylcholine (4:1). The half-time for photolabeling was approximately 7 min consistent with the half-time of 9 min for photolysis of TID under our conditions. Comparable amounts of TID per mg of protein were incorporated into each subunit. Purified sodium channels reconstituted in phosphatidylcholine vesicles were also labeled by TID with comparable incorporation per mg of protein into all three subunits. The efficiency of photolabeling of the three subunits was reduced from 39 to 44% by a 2-fold expansion of the hydrophobic phase of the reaction mixture but was unaffected by a 2-fold expansion of the aqueous phase, confirming that the photolabeling reaction took place in the lipid phase of the vesicle bilayer. The hydrophobic properties of the sodium channel subunits were examined further using phase separation in the nonionic detergent Triton X-114. Under conditions in which beta 1 is dissociated from alpha, the beta 1 subunit was preferentially extracted into the Triton X-114 phase, and the disulfide-linked alpha beta 2 complex was retained in the aqueous phase. When the disulfide bonds between the alpha and beta 2 subunits were reduced with dithioerythritol, the beta 2 subunit was also preferentially extracted into the Triton X-100 phase leaving the free alpha subunit in the aqueous phase. A preparative method for isolation of the beta 1 and beta 2 subunits was developed based on this technique. Considered together, the results of our hydrophobic labeling and phase separation experiments indicate that the alpha, beta 1, and beta 2 subunits all have substantial hydrophobic domains that may interact with the hydrocarbon phase of phospholipid bilayer membranes. Since the alpha subunit is known to be a transmembrane protein with many potential membrane-spanning segments, we conclude that the beta 1 and beta 2 subunits are likely to also be integral membrane proteins with one or more membrane-spanning segments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.     

Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: studies with antibodies against the alpha subunits

Polyclonal antibodies (PAC-2) against the purified skeletal muscle calcium channel were prepared and shown to be directed against alpha subunits of this protein by immunoblotting and immunoprecipitation. These polypeptides have an apparent molecular weight of 162,000 without reduction of disulfide bonds. Under conditions where the functional properties of the purified skeletal muscle calcium channel are retained, beta subunits (Mr 50,000) and gamma subunits (Mr 33,000) are coprecipitated, demonstrating specific noncovalent association of these three polypeptides in the purified skeletal muscle channel. PAC-2 immunoprecipitated cardiac calcium channels labeled with [3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- (methoxycarbonyl)pyridine-3-carboxylate ([3H]PN200-110) at a 3-fold higher concentration than skeletal muscle channels. Preincubation with cardiac calcium channels blocked only 49% of the immunoreactivity of PAC-2 toward skeletal muscle channels, indicating that these two proteins have both homologous and distinct epitopes. The immunoreactive component of the cardiac calcium channel was identified by immunoprecipitation and polyacrylamide gel electrophoresis as a polypeptide with an apparent molecular weight of 170,000 before reduction of disulfide bonds and 141,000 after reduction, in close analogy with the properties of the alpha 2 subunits of the skeletal muscle channel. It is concluded that these two calcium channels have a homologous, but distinct, alpha subunit as a major polypeptide component.     

The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

Subunits of purified calcium channels. Alpha 2 and delta are encoded by the same gene

Polyacrylamide gel electrophoresis of purified rabbit skeletal muscle L-type calcium channel before and after reduction of disulfide bonds confirmed that 27- and 24-kDa forms of the delta subunit are disulfide-linked to the 143-kDa alpha 2 subunit. The amino acid sequences of three peptides obtained by tryptic digestion of the delta subunits corresponded to amino acid sequences predicted from the 3' region of the mRNA encoding alpha 2. One of these peptides had the same sequence as the N terminus of the 24- and 27-kDa forms of the delta subunit and corresponded to residues 935-946 of the predicted alpha 2 primary sequence. Anti-peptide antibodies directed to regions on the N-terminal side of this site recognized the 143-kDa alpha 2 subunit in immunoblots of purified calcium channels under reducing conditions, whereas an antipeptide antibody directed toward a sequence on the C-terminal side of this site recognized 24- and 27-kDa forms of the delta subunit. A similar result was obtained after immunoblotting using purified transverse tubules or crude microsomal membrane preparations indicating that alpha 2 and delta occur as distinct disulfide-linked polypeptides in skeletal muscle membranes. Thus, the delta subunits are encoded by the same gene as the alpha 2 subunit and are integral components of the skeletal muscle calcium channel.     

A monoclonal antibody to the beta subunit of the skeletal muscle dihydropyridine receptor immunoprecipitates the brain omega-conotoxin GVIA receptor.

Antibodies against the subunits of the dihydropyridine-sensitive L-type calcium channel of skeletal muscle were tested for their ability to immunoprecipitate the high affinity (Kd = 0.13 nM) 125I-omega-conotoxin GVIA receptor from rabbit brain membranes. Monoclonal antibody VD2(1) against the beta subunit of the dihydropyridine receptor from skeletal muscle specifically immunoprecipitated up to 86% of the 125I-omega-conotoxin receptor solubilized from brain membranes whereas specific antibodies against the alpha 1, alpha 2, and gamma subunits did not precipitate the brain receptor. Purified skeletal muscle dihydropyridine receptor inhibited the immunoprecipitation of the brain omega-conotoxin receptor by monoclonal antibody VD2(1). The dihydropyridine receptor from rabbit brain membranes was also precipitated by monoclonal antibody VD2(1). However, neither the neuronal ryanodine receptor nor the sodium channel was precipitated by monoclonal antibody VD2(1). The omega-conotoxin receptor immunoprecipitated by monoclonal antibody VD2(1) showed high affinity 125I-omega-conotoxin binding, which was inhibited by unlabeled omega-contoxin and by CaCl2 but not by nitrendipine or by diltiazem. An antibody against the beta subunit of the skeletal muscle dihydropyridine receptor stained 58- and 78-kDa proteins on immunoblot of the omega-conotoxin receptor, partially purified through heparin-agarose chromatography and VD2(1)-Sepharose chromatography. These results suggest that the brain omega-conotoxin-sensitive calcium channel contains a component homologous to the beta subunit of the dihydropyridine-sensitive calcium channel of skeletal muscle and brain.     

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.     

Stabilization of a sodium channel state with high affinity for saxitoxin by intramolecular cross-linking. Evidence for allosteric effects of saxitoxin binding

Incubation of purified rat brain sodium channels at 37 degrees C or at high ionic strength causes a concomitant loss of saxitoxin-binding activity and dissociation of beta 1 subunits. Reaction with hydrophilic carbodiimides produced a resistance against the loss of saxitoxin binding and caused covalent cross-linking of alpha, beta 1, and beta 2 subunits. In the presence of saxitoxin, this cross-linking reaction led to formation of a state with increased affinity for saxitoxin. However, analysis of the concentration dependence of covalent cross-linking and its inhibition by hydrophilic nucleophiles showed that the stabilization of the saxitoxin-binding activity was due to the formation of a small number of isopeptide bonds in the alpha subunit rather than to cross-linking of alpha and beta 1 subunits. In the presence of amine nucleophiles, carbodiimides caused loss of saxitoxin binding, which was prevented in the presence of the toxin. Nucleophiles yielding positively charged amide products were more effective than those forming uncharged or negatively charged products. Under conditions where saxitoxin protected the binding activity of the sodium channel from inactivation, the overall availability of carboxyl groups for reaction was increased, providing evidence for a toxin-induced conformational change on binding. These results are considered in terms of an allosteric model of saxitoxin binding, in which the functional form of the sodium channel having high affinity for saxitoxin can be stabilized against inactivation by noncovalent interactions with beta 1 subunits, binding of saxitoxin and tetrodotoxin, or intramolecular cross-linking of amino acid residues within the alpha subunit.     

Monoclonal antibodies to receptors for insulin and somatomedin-C

Three monoclonal antibodies, designated alpha IR-1, alpha IR-2, and alpha IR-3, were prepared by fusing FO myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of insulin receptors from human placenta. These antibodies were characterized by their ability to immunoprecipitate solubilized receptors labeled with 125I-insulin or 125I-somatomedin-C in the presence or absence of various concentrations of unlabeled insulin or somatomedin-C. alpha IR-1 preferentially immunoprecipitates insulin receptors and also less effectively immunoprecipitates somatomedin-C receptors, while alpha IR-2 and alph IR-3 preferentially immunoprecipitate somatomedin-C receptors, but may also weakly immunoprecipitate insulin receptors. These three monoclonal antibodies, as well as A410, a rabbit polyclonal antibody, were used to immunoprecipitate insulin and somatomedin-C receptors from solubilized human lymphoid (IM-9) cells and human placenta membranes that had been 125I-labeled with lactoperoxidase. Analysis of the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both receptors are composed of alpha and beta subunits. The beta subunit of the insulin receptor (immunoprecipitated by alpha IR-1 and A410) has a slightly more rapid mobility than the corresponding subunit of the somatomedin-C receptor (immunoprecipitated by alpha IR-2 and alpha IR-3). Interestingly, the alpha subunit of the placenta somatomedin-C receptor has a slightly faster mobility than its counterpart from IM-9 cells. Immunoprecipitation of receptor that had been reduced and denatured to generate isolated subunits indicates that alpha IR-2 and alpha IR-3 interact with the alpha subunit of the somatomedin-C receptor while A410 interacts with both subunits of the insulin receptor. alpha IR-1 failed to react with reduced and denatured receptors.     

Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord, and retina

Monoclonal antibodies that recognize the alpha 2 delta subunits of calcium channels from skeletal muscle immunoprecipitate a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. They also immunoprecipitate 64% of rabbit brain dihydropyridine-sensitive calcium channels followed by immunoprecipitation reveals alpha 1-, alpha 2 delta-, and beta-like subunits that have apparent molecular masses of 175, 142, and 57 kd, respectively. A polypeptide of 100 kd is also specifically immunoprecipitated. Immunocytochemical studies identify dihydropyridine-sensitive calcium channels in neuronal somata and proximal dendrites in rat brain, spinal cord, and retina. Staining of many neuronal somata is uneven, revealing relatively high densities of dihydropyridine-sensitive calcium channels at the base of major dendrites. L-type calcium channels in this location may serve to mediate long-lasting increases in intracellular calcium in the cell body in response to excitatory inputs to the dendrites.     

Subunit structure and dynamics of the insulin receptor

A model for the minimum subunit composition and stiochiometry of the physiologically relevant insulin receptor has been deduced based on results obtained by affinity labeling of this receptor in a variety of cell types and species. We propose that the receptor is a symmetrical disulfide-linked heterotetramer composed of two alpha (apparent Mr = 125,000) and two beta (apparent Mr = 90,000) glycoprotein subunits in the configuration (beta-S-S-alpha)-S-S-(alpha-S-S-beta). The disulfide or disulfides linking the two (alpha-S-S-beta) halves (class I disulfides) exhibit greater sensitivity to reduction by exogenous reductants than those linking the alpha and beta subunits (class II disulfides). When the class I disulfides are reduced by addition of diothiothreitol to intact cells, the receptor retains its ability to bind insulin and to effect a biological response. The beta subunit contains a site at about the center of its amino acid sequence that is extremely sensitive to proteolytic cleavage by elastaselike proteases, yielding a beta 1 fragment (Mr = 45,000) that remains disulfide linked to the receptor complex and a free beta 2 fragment. Binding of insulin to the receptor complex appears to result in the formation or stabilization of a new receptor conformation as evidenced by an altered susceptibility of the alpha subunit to exogenous trypsin. A receptor structure with high affinity for insulinlike growth factor (IGF) I and low affinity for insulin in fibroblast and placental membranes has also been affinity labeled. It exhibits the same structural features found for the insulin receptor, including two classes of disulfide bridges and beta subunits highly sensitive to proteolytic cleavage. These recent observations identifying the presence of distinct insulin and IGF-I receptors that share similar complex structures suggest that these hormones may also share common mechanisms of transmembrane signaling.     

Structural characterization of insulin receptors. II. Subunit composition of receptors from turkey erythrocytes

In the preceding paper (Aiyer, R. A. (1983) J. Biol. Chem. 258, 14992-14999), the hydrodynamic properties of insulin receptors from turkey erythrocyte plasma membranes solubilized in nondenaturing detergents (Triton X-100 and sodium deoxycholate) were characterized. Two specific insulin-binding species are observed after velocity sedimentation in linear sucrose density gradients: peak II whose protein molecular weight (Mp) is 180,000 +/- 45,000 and its disulfide-linked dimer, peak I (Mp, 355,000 +/- 65,000). This paper describes the subunit composition of these species determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Insulin receptors were covalently attached to [125I]iodoinsulin with disuccinimidyl suberate. After solubilization in Triton X-100 or deoxycholate, peaks I and II were separated by sedimentation and subjected to SDS-PAGE; the constituent polypeptides were then identified by autoradiography. Under reducing conditions, both peaks I and II yield a major band of apparent molecular weight (Mapp) of 135,000; this band most likely represents the insulin-binding subunit (alpha). Minor bands of lower molecular weight are also seen whose significance is not entirely obvious. Under nonreducing conditions, peak I yields bands at Mapp = 230,000 and at greater than 240,000, while peak II yields bands at Mapp = 120,000 and 200,000. When these bands were cut out of the gel and subjected to SDS-PAGE following reduction with 10% beta-mercaptoethanol, all of them produced a single band that migrated with Mapp = 135,000. These results indicate that the alpha subunit is linked by disulfide bonds to at least one more subunit (beta). It is also apparent that the alpha subunit travels with higher mobility (Mapp = 120,000) under nonreducing conditions, suggesting the presence of intrachain disulfide bonds. Thus, peak II has a minimum subunit composition of alpha beta, where alpha is the insulin-binding subunit with a minimum Mr = 120,000-135,000 and beta has a minimum Mr = 80,000-90,000. And peak I, the disulfide-linked dimer of peak II, has a minimum subunit composition of alpha 2 beta 2. These results were further confirmed by cross-linking of protein subunits with glutaraldehyde, an (alpha, omega)-dialdehyde that reacts with amino groups. Within the limits of error, these molecular weights are in agreement with those estimated from the hydrodynamic properties of the detergent-solubilized, native receptor species reported in the preceding paper.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.