Putative gamma-subunit of the IL-2 receptor is detected in low, intermediate, and high affinity IL-2 receptor-bearing cells

Three forms of the IL-2R have been reported: low, intermediate, and high affinity. Each form correlates with the expression of two different chains: p55 (alpha-subunit) and p75 (beta-subunit). We report here a putative new subunit of the IL-2R, termed p95-110 or gamma-subunit. This new subunit has a molecular mass of 95 to 110 kDa and is expressed in low, intermediate, and high affinity IL-2R-bearing cells. We propose that p95-110 is the partner of p75 in the formation of intermediate affinity IL-2R, inasmuch as neither p75 nor p95-110 alone can bind IL-2.     

Three distinct classes of the alpha-subunit of the nuclear pore-targeting complex (importin-alpha) are differentially expressed in adult mouse tissues.

The process of active nuclear protein transport is mediated by the nuclear localization signal (NLS). An NLS-containing karyophile forms a stable complex, termed the nuclear pore-targeting complex, to target nuclear pores. The alpha-subunit of the complex (importin-alpha) binds to the NLS and the beta-subunit (importin-beta) carries the alpha-subunit, bound to the NLS substrate, into the nucleus. To date, five mouse alpha-subunits have been identified and classified into three subfamilies (alpha-P, alpha-Q, and alpha-S). The expression of these alpha-subunits and the beta-subunit in various adult mouse tissues was examined by immunoblotting and immunohistochemistry using antibodies specific for each subfamily of the alpha-subunit or the beta-subunit. The beta-subunit was found to be ubiquitously expressed, whereas each subfamily of the alpha-subunit showed a unique expression pattern in various tissues, especially in brain and testis. In brain, the expression of alpha-P was not observed, whereas alpha-S was significantly expressed in Purkinje cells, and pyramidal cells of the hippocampus and cerebral cortex. In testis, alpha-P was expressed predominantly in primary spermatocytes, whereas alpha-Q was found mainly in Leydig cells. Expression of alpha-S was detected in almost all cells in convoluted seminiferous tubules and Leydig cells to a similar extent. These results suggest that nuclear protein import may be controlled in a tissue-specific manner by alpha-subunit family proteins.     

Expression of the human IL-2 receptor on lymphocytes involves rapid turnover of its p55 alpha-subunit (Tac)

Upon mitogenic stimulation, both mRNA encoding the p55 alpha-subunit (Tac) of the human IL-2R alpha and IL-2R alpha protein are induced and expressed in tonsil lymphocyte populations over several days. Using a quantitative dot-blot immunoassay for the IL-2R alpha subunit, a rapid disappearance of this polypeptide from cells is demonstrated in the presence of the translation inhibitor, cycloheximide. The half-life of IL-2R alpha subunit protein is 2 to 3 h. This decline in cell-associated IL-2R alpha subunit is matched by a rapid decline in IL-2R alpha on the cell surface and is not accompanied by any increase in soluble IL-2R alpha protein. Long term expression of the IL-2R on the cell surface is thus the result of continual synthesis and rapid breakdown of IL-2R alpha chains in the cell. Steady state expression of the IL-2R after an immune stimulus hence depends upon continuous expression of the IL-2R alpha subunit gene. Rapid turnover of the unstable alpha-subunit on the cell surface provides a novel mechanism for sensitive control of functional IL-2R expression.     

Immunohistochemical localization of S-100 protein subunits (alpha and beta) in dorsal root ganglia of the rat

The distribution of S-100 protein and their subunits (alpha and beta) in lumbar dorsal root ganglia of adult rat was investigated immunohistochemically using monoclonal antibodies against the S-100 protein, alpha-subunit and beta-subunit of S-100 protein. The conventional S-100 protein antibody stained both neurons (large and intermediate in size; 20.3% and 41 +/- 3.2 microns of diameter) and glial cells (satellite cells and Schwann cells). The immunoreaction for the alpha-subunit was observed in the perikarya of some large and intermediate sized neurons (17.2%, 45.6 +/- 6.1 microns of diameter), satellite cells and Schwann cells, whereas the beta-subunit immunoreactivity was found principally in glial cells, and in a scarce number of large and intermediate sized neurons (2.8%, 43.3 +/- 5 microns of diameter) Our results demonstrate that a subpopulation of large and intermediate sized neurons of lumbar DRG contain alpha- and beta-subunits of S-100 protein, being alpha-subunit predominant. Furthermore, the satellite glial and Schwann cells contain also the two subunits but mainly beta-subunit. These data confirm previous studies about the presence of S-100 protein in neurons of the central and peripheral nervous system.     

Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells.

PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation. Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells. Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM). Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP. By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells. Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate. These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.     

Down-regulation of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition

Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the alpha- and/or beta-subunit or arises from a physical dissociation of the alpha-subunit from the beta-subunit. The beta-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using alpha- and beta-subunit-specific antibodies, detected bands of 110-125 and 36 kDa, respectively. Protein expression levels of the alpha-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, alpha-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased beta-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the alpha- and beta-subunits in samples taken before or after the onset of labor. We conclude that during labor, the alpha-subunit is not physically uncoupled from the beta-subunit, but a decline occurs in the level of beta-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced beta-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.     

Hormonotoxins. I. Strategy for synthesis of ovine luteinizing hormone--gelonin conjugate bearing the toxin in the beta-subunit

The amino groups in the beta-subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N-succinimidyl-3-(2-pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified beta-subunit was able to hybridize with free LH alpha-subunit and the complex retained full biological activity. However, when gelonin was coupled to the beta-subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free alpha-subunit. This has important implications for the design in the synthesis of gonadotropin-toxin/drug conjugates.     

Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole

Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.     

Antipeptide antibodies toward the extracellular domain of insulin receptor beta-subunit

In order to investigate structure and function of beta-subunit extracellular portion, four polyclonal antibodies (AP1, AP2, AP3 and AP4) toward peptides comprised in this region were generated. None of them recognizes native human and rat insulin receptor both in vitro and in whole cells. Two antibodies, AP1 and AP2, immunoprecipitate isolated (DTT-reduced) human beta-subunits and bind to human IM-9 cell after alpha-subunit tryptic cleavage. Only AP1 recognizes rat beta-subunit both in vitro and in trypsin treated rat FAD cells. These findings suggest that: (i) the extracellular portion of the insulin receptor beta-subunit is partially covered by the alpha-subunit in human and rat native insulin receptors; (ii) human and rat beta-subunit extracellular domains are different, at least in the amino acid sequence corresponding to residues 785-796 of the human insulin receptor.     

The carboxyl-terminal 161 amino acids of the Na,K-ATPase alpha-subunit are sufficient for assembly with the beta-subunit.

Chimeric cDNAs encoding regions of the Na,K-ATPase alpha-subunit and a sarcoplasmic reticulum Ca(2+)-ATPase were constructed and expressed together with the avian Na,K-ATPase beta-subunit cDNA in COS-1 cells to determine which regions of the alpha-subunit are required for assembly with the beta-subunit. Assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the avian beta-subunit. A chimera composed of the amino-terminal two-thirds of the Na,K-ATPase and carboxyl-terminal one-third of the Ca(2+)-ATPase did not assemble with the avian beta-subunit. In contrast, the reciprocal chimera, containing the carboxyl-terminal one-third of the Na,K-ATPase, assembled with the beta-subunit. A third chimera, in which 161 amino acids of the Na,K-ATPase carboxyl terminus replaced the corresponding amino acids of the Ca(2+)-ATPase carboxyl terminus, also assembled with the beta-subunit. These results suggest that the aminoacyl residues of the Na,K-ATPase alpha-subunit critical for subunit assembly lie within the carboxyl-terminal 16% of the sequence.     

Hormonotoxins. Effects of modifying the gonadotropin alpha-subunit on the generation of lutropin-toxin conjugates

On the basis of the observation that in the hormonotoxin oLH-gelonin conjugation of the toxin occurs via the alpha-subunit of lutropin, an attempt was made to develop a general method for generation of similar hybrid proteins involving other glycoprotein hormones. In this approach sites suitable for conjugation would be first introduced into the alpha-subunit, hybridized with any native hormone specific beta-subunit of choice (LH-beta, FSH-beta, TSH-beta) following which the toxic component gelonin would be added on in the form of gelonin-S-S-alpha---beta complex. Thus, thiolated lutropin alpha-subunit recombined well with free lutropin beta-subunit, yielding a hybrid which was active in terms of receptor binding, immunoreactivity, and steroidogenic properties. However, subsequent conjugation with thiolated gelonin, a ribosome inactivating protein, resulted in dissociation of beta-subunit from its non-covalent union with the thiolated alpha-subunit. It is concluded that the addition of positively charged gelonin at particular sites on the alpha-subunit led to the destabilization of the lutropin quaternary structure.     

A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits

The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.     

A fusion protein of the gp130 and interleukin-6Ralpha ligand-binding domains acts as a potent interleukin-6 inhibitor

Interleukin (IL)-6 is involved in the maintenance and progression of several diseases such as multiple myeloma, rheumatoid arthritis, or osteoporosis. The present work aims at the development of an IL-6 inhibitor for the use in anti-cytokine therapies. The IL-6 receptor is composed of two different subunits, an alpha-subunit (IL-6Ralpha) that binds IL-6 with low affinity and a beta-subunit (gp130) that binds the IL-6.IL-6Ralpha complex with high affinity and as a result triggers intracellular signaling. In its soluble form, gp130 is a natural antagonist that neutralizes IL-6.soluble IL-6Ralpha complexes. It was our strategy to appropriately fuse the two receptor subunit fragments involved in IL-6 receptor complex formation to bind IL-6 with high affinity and to antagonize its effects. The ligand-binding domains of gp130 (D1-D2-D3) and IL-6Ralpha (D2-D3) were connected using three different linkers. The resulting constructs were expressed in stably transfected insect cells and tested for their ability to inhibit IL-6 activity in several in vitro systems. All fusion proteins were strong inhibitors of IL-6 signaling and abrogated IL-6-induced phosphorylation of STAT3, proliferation of transfected Ba/F3 cells, and induction of acute-phase protein synthesis. As intended, the fused receptors were much more effective than the separately expressed soluble receptor proteins. The fusion protein strategy presented here can also be applied to other cytokines that signal via receptors composed of two different subunits to design new potent inhibitors for anti-cytokine therapies.     

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.     

Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ER to reach to the cell surface as a functional holoenzyme. We studied the quantity control mechanism of the H(+),K(+)-ATPase in the ER by using a heterologous expression system in human embryonic kidney 293 cells. The alpha-subunit in the alpha-expressing cells was degraded more rapidly than in the alpha+beta-expressing cells. It was stabilized, however, in the presence of a proteasome inhibitor, lactacystin. Polyubiquitination of the alpha-subunit was observed in the alpha-expressing cells as well as in the alpha+beta-expressing cells. The extent of polyubiquitination was higher in the former alpha-expressing cells especially in the presence of lactacystin. On the other hand, polyubiquitination of the beta-subunit was not observed in the absence and presence of lactacystin. When the alpha-subunit was coexpressed with a mutant beta-subunit that lacks alpha/beta assembly capacity, degradation of the alpha-subunit was accelerated in parallel with increased polyubiquitination of the alpha-subunit. These results indicate that the ubiquitin/proteasome system is involved in degradation of the unassembled alpha-subunits in the ER to control the cell surface expression of the functional alpha/beta holoenzymes.     

The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.     

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its beta-subunit that has been likened to a "seatbelt" because it surrounds alpha-subunit loop 2 and its end is "latched" by an intrasubunit disulfide bond to the beta-subunit core. As shown here, assembly begins when parts of the NH(2) terminus, cysteine knot, and loops 1 and 3 of the alpha-subunit dock reversibly with parts of the NH(2) terminus, cystine knot, and loop 2 of the hCG beta-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of alpha-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a "zipper-like" sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the beta-subunit cystine knot, alpha-subunit loop 2, and the small seatbelt loop. The resulting intersubunit beta-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.     

Limited Proteolysis of Soybean Beta-conglycinin

The tryptic digestion of beta-conglycinin was studied by the pH-stat method and sodium dodecyl sulfate gel electrophoresis. The proteolysis of the protein was not affected by the change of ionic strength. This property was distinct from that of glycinin which is degraded rapidly at a low ionic strength.

Five stable fragments were generated in the degradation course. Two kinds of beta-conglycinin, one of which consisted of only the beta-subunit and the other of only the alpha’ and alpha-subunit, were isolated to elucidate the original subunit of the fragments. Two fragments (AT-1 and AT-2) from the alpha’ and alpha-subunit, and three fragments (BT-2-BT-4) from the beta-subunit were generated. The molecular weights of the fragments were similar to those of the glycinin fragments. From the fragment pattern of the beta-subunit, the presence of two types of the beta-subunit was expected.