Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ～65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.     

Channel gating of the glycine receptor changes accessibility to residues implicated in receptor potentiation by alcohols and anesthetics

The glycine receptor is a target for both alcohols and anesthetics, and certain amino acids in the alpha1 subunit transmembrane segments (TM) are critical for drug effects. Introducing larger amino acids at these positions increases the potency of glycine, suggesting that introducing larger residues, or drug molecules, into the drug-binding cavity facilitates channel opening. A possible mechanism for these actions is that the volume of the cavity expands and contracts during channel opening and closing. To investigate this hypothesis, mutations for amino acids in TM1 (I229C) and TM2 (G256C, T259C, V260C, M263C, T264C, S267C, S270C) and TM3 (A288C) were individually expressed in Xenopus laevis oocytes. The ability of sulfhydryl-specific alkyl methanethiosulfonate (MTS) compounds of different lengths to covalently react with introduced cysteines in both the closed and open states of the receptor was determined. S267C was accessible to short chain (C3-C8) MTS in both open and closed states, but was only accessible to longer chain (C10-C16) MTS compounds in the open state. Reaction with S267C was faster in the open state. I229C and A288C showed state-dependent reaction with MTS only in the presence of agonist. M263C and S270C were also accessible to MTS labeling. Mutated residues more intracellular than M263C did not react, indicating a floor of the cavity. These data demonstrate that the conformational changes accompanying channel gating increase accessibility to amino acids critical for drug action in TM1, TM2, and TM3, which may provide a mechanism by which alcohols and anesthetics can act on glycine (and likely other) receptors.     

HbA1c-based diabetes diagnosis among patients with glucokinase mutation (GCK-MODY) is affected by a genetic variant of glucose-6-phosphatase (G6PC2)

Aims Genetic variation at the rs560887 locus of the glucose-6-phosphatase, catalytic?2 gene (G6PC2) is known to affect regulation of fasting glycaemia. We determined the rs560887 genotype of patients with monogenic diabetes and glucokinase gene mutations (GCK-MODY) and correlated the genotypes with HbA(1c) levels. Methods Patients from families with GCK-MODY were recruited from two large cohorts from Poland (n?=?128) and the Czech Republic (n?=?154). Genotypes at the rs560887 polymorphic site in G6PC2 were examined using real-time quantitative polymerase chain reaction. The effect of rs560887 genotype on age at diagnosis of GCK-MODY and initial HbA(1c) levels were evaluated separately within both cohorts. Following that, a meta-analysis of rs560887 genotype-HbA(1c) associations of both Polish and Czech cohorts was performed to confirm homogeneity of findings and validate cohort-specific results. Results GG homozygosity at rs560887 was associated with marginally elevated HbA(1c) levels (P?=?0.07 in both cohorts). The effects observed in both groups were very homogeneous (Q?=?0.18; P?=?0.68). Meta-analysis showed that GG homozygosity at rs560887 was associated with mean HbA(1c) levels higher by 2.4?mmol/mol (0.24%), 95%?CI 0.5-4.4?mmol/mol (0.05-0.44%) than in individuals with other genotypes. Additionally, meta-analysis of both cohorts showed that GG homozygous individuals had higher odds of reaching the 48?mmol/mol (6.5%) diagnostic threshold of diabetes; (odds ratio 1.90; 95%?CI 1.07-3.36; P?=?0.03). No such effects were observed for age at diagnosis of diabetes. Conclusions Variation at the rs560887 locus of G6PC2 is associated with worse glycated haemoglobin levels in individuals with GCK mutations; GG homozygotes are more likely to meet diagnostic criteria for diabetes based on HbA(1c) level.     

Association with nitric oxide synthase on insulin secretory granules regulates glucokinase protein levels

Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect β-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in βTC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.     

Mutations in sialidosis impair sialidase binding to the lysosomal multienzyme complex

Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the catabolism of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (sialidosis type II). We analyzed the effect of the missense mutations G68V, S182G, G227R, F260Y, L270F, A298V, G328S, and L363P, which are identified in the sialidosis type I and sialidosis type II patients, on the activity, stability, and intracellular distribution of sialidase. We found that three mutations, F260Y, L270F, and A298V, which are clustered in the same region on the surface of the sialidase molecule, dramatically reduced the enzyme activity and caused a rapid intralysosomal degradation of the expressed protein. We suggested that this region might be involved in sialidase binding with lysosomal cathepsin A and/or beta-galactosidase in the multienzyme lysosomal complex required for the expression of sialidase activity. Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts confirmed this hypothesis and showed that sialidase deficiency in some sialidosis patients results from disruption of the lysosomal multienzyme complex.     

Characterization of the molecular motions of constitutively active G protein-coupled receptors for parathyroid hormone

The molecular mechanism of constitutive activity of the G protein-coupled receptor for human parathyroid hormone (PTH1) has been examined by molecular dynamics (MD) simulations. The single point mutations H223R, T410P, and I458R, of the PTH1 receptor result in ligand-independent receptor activation. Extensive MD simulations indicate that each of the mutations, through different mechanisms, lead to very similar conformational changes of the third intracellular loop. The structural changes, centered on K405 in the C-terminus of the third intracellular loop, can be traced back to the single-point mutations by calculation of the forces and torques responsible for the collective motions of the receptor. This analysis indicates a direct correlation between the conformational preferences of the cytoplasmic loop and the mutations in different locations of the receptor: TM2 (H223R), TM6 (T410P), and TM7 (1458R). Given the pivotal role of the third intracellular loop of PTH1 in coupling to the G proteins, the structural changes induced by these single-point mutations may be responsible for the ligand-free activation of the receptor. These results coupled with the high-resolution structure of the third cytoplasmic loop of PTH1, previously determined in our laboratory, provide unique insight into the mechanism of ligand free activation of the PTH1 receptor.     

YscU cleavage and the assembly of Yersinia type III secretion machine complexes

YscU, a component of the Yersinia type III secretion machine, promotes auto-cleavage at asparagine 263 (N263). Mutants with an alanine substitution at yscU codon 263 displayed secretion defects for some substrates (LcrV, YopB and YopD); however, transport of effector proteins into host cells (YopE, YopH, YopM) continued to occur. Two yscU mutations were isolated that, unlike N263A , completely abolished type III secretion; YscU_G127D promoted auto-cleavage at N263, whereas YscU_G270N did not. When fused to glutathione S-transferase (Gst), the YscU C-terminal cytoplasmic domain promoted auto-cleavage and Gst-YscU_C also exerted a dominant-negative phenotype by blocking type III secretion. Gst–YscU_C/N263A caused a similar blockade and Gst–YscU_C/G270N reduced secretion. Gst–YscU_C and Gst–YscU_C/N263A bound YscL, the regulator of the ATPase YscN, whereas Gst–YscU_C/G270N did not. When isolated from Yersinia , Gst–YscU_C and Gst–YscU_C/N263A associated with YscK–YscL–YscQ; however, Gst–YscU_C/G270N interacted predominantly with the machine component YscO, but not with YscK–YscL–YscQ. A model is proposed whereby YscU auto-cleavage promotes interaction with YscL and recruitment of ATPase complexes that initiate type III secretion.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.     

Direct Activation of Mitogen-Activated Protein Kinase Kinase Kinase MEKK1 by the Ste20p Homologue GCK and the Adapter Protein TRAF2

Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.     

Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase

Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence.Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning.In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.     

Caspase proteolysis of desmin produces a dominant-negative inhibitor of intermediate filaments and promotes apoptosis

Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD downward arrow M(264)) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of desmin at Asp(263) has important functional consequences, including the production of an amino-terminal cleavage product, N-desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a dominant-negative inhibitor of filament assembly, both for desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant desmin D263E mutant partially protects cells from tumor necrosis factor-alpha-induced apoptosis. Taken together, these results indicate that caspase proteolysis of desmin at Asp(263) produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.     

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.     

Screening of mutations and polymorphisms in the glucokinase gene in Czech diabetic and healthy control populations

Glucokinase (GCK) plays a key role in glucose metabolism. GCK mutations are known as a pathogenic cause of maturity-onset diabetes of the young type 2 (MODY2). These mutations are also found in gestational diabetics. The aim of our study was to assess the variability of the GCK gene in the Czech diabetic and control populations. We screened all 10 exons specific for the pancreatic isoform of glucokinase (1a and 2-10) including the intron flanking regions in 722 subjects (in 12 patients with an unrecognised type of MODY and their 10 family members, 313 patients with diabetes mellitus type 2 (DM2), 141 gestational diabetics (GDM), 130 healthy offspring of diabetic parents, and 116 healthy controls without family history of DM2). In two MODY families we identified two mutations in exon 2 of the GCK gene: a novel mutation Val33Ala and the previously described mutation Glu40Lys. In other subgroups (excluding MODY families) we detected only intronic variants and previously described polymorphisms in exons 6 (Tyr215Tyr) and 7 (Ser263Ser), we did not find any known GCK pathogenic mutation. We observed no difference in the frequencies of GCK polymorphisms between Czech diabetic (DM2, GDM) and non-diabetic populations.     

Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin binding.

The mu opioid receptor (MOR) plays a central role in mediating acute and chronic effects of narcotic drugs. Three rare single nucleotide polymorphisms in the hMOR gene have been identified that cause amino acid substitutions in the third intracellular (i3) loop of MOR (R260H, R265H, and S268P). Genotyping 252 individuals of the Coriell collection identified one allele encoding the R265H-MOR variant and a new variant encoding D274N-MOR. Variants R260H-, R265H-, and S268P-MOR were constructed and transfected into HEK293 cells. Morphine stimulated G protein coupling of the three receptor variants to a maximal level approaching that of wild type MOR. In contrast, spontaneous, agonist-independent (basal) MOR signaling, proposed to play a role in opioid tolerance and dependence, was significantly reduced for R260H- and R265H-MOR. Moreover, domains within the i3 loop of MOR have been shown to interact with both G proteins and calmodulin (CaM). CaM binding was deficient for variants R265H- and S268P-MOR, suggesting that domains for G protein coupling and CaM binding overlap partially. Morphine pretreatment significantly enhanced basal G protein coupling of wild type MOR, which is thought to result from release of CaM. In contrast basal G protein coupling activity of the three variants was unaffected by morphine pretreatment consistent with diminished CaM regulation, low basal activity, or both. In conclusion, each of the three single nucleotide polymorphisms mapping to the i3 loop of MOR caused substantial changes in basal G protein coupling, CaM binding, or both. Carriers of the mutant alleles might display altered responses to narcotic analgesics.     

Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft

Penicillin G acylase is a periplasmic protein, cytoplasmically expressed as a precursor polypeptide comprising a signal sequence, the A and B chains of the mature enzyme (209 and 557 residues respectively) joined by a spacer peptide of 54 amino acid residues. The wild-type AB heterodimer is produced by proteolytic removal of this spacer in the periplasm. The first step in processing is believed to be autocatalytic hydrolysis of the peptide bond between the C-terminal residue of the spacer and the active-site serine residue at the N terminus of the B chain. We have determined the crystal structure of a slowly processing precursor mutant (Thr263Gly) of penicillin G acylase from Escherichia coli, which reveals that the spacer peptide blocks the entrance to the active-site cleft consistent with an autocatalytic mechanism of maturation. In this mutant precursor there is, however, an unexpected cleavage at a site four residues from the active-site serine residue. Analyses of the stereochemistry of the 260-261 bond seen to be cleaved in this precursor structure and of the 263-264 peptide bond have suggested factors that may govern the autocatalytic mechanism.     

Beta2-adrenergic receptor lysosomal trafficking is regulated by ubiquitination of lysyl residues in two distinct receptor domains

Agonist stimulation of the β2-adrenergic receptors (β2ARs) leads to their ubiquitination and lysosomal degradation. Inhibition of lysosomal proteases results in the stabilization and retention of internalized full-length β2ARs in the lysosomes, whereas inhibition of proteasomal proteases stabilizes newly synthesized β2ARs in nonlysosomal compartments. Additionally, a lysine-less β2AR (0K-β2AR) that is deficient in ubiquitination and degradation is not sorted to lysosomes unlike the WT β2AR, which is sorted to lysosomes. Thus, lysosomes are the primary sites for the degradation of agonist-activated, ubiquitinated β2ARs. To identify the specific site(s) of ubiquitination required for lysosomal sorting of the β2AR, four mutants, with lysines only in one intracellular domain, namely, loop 1, loop 2, loop 3, and carboxyl tail were generated. All of these receptor mutants coupled to G proteins, recruited β-arrestin2, and internalized just as the WT β2AR. However, only loop 3 and carboxyl tail β2ARs with lysines in the third intracellular loop or in the carboxyl tail were ubiquitinated and sorted for lysosomal degradation. As a complementary approach, we performed MS-based proteomic analyses to directly identify ubiquitination sites within the β2AR. We overexpressed and purified the β2AR from HEK-293 cells with or without prior agonist exposure and subjected trypsin-cleaved β2AR to LC-MS/MS analyses. We identified ubiquitinated lysines in the third intracellular loop (Lys-263 and Lys-270) and in the carboxyl tail (Lys-348, Lys-372, and Lys-375) of the β2AR. These findings introduce a new concept that two distinct domains in the β2AR are involved in ubiquitination and lysosomal degradation, contrary to the generalization that such regulatory mechanisms occur mainly at the carboxyl tails of GPCRs and other transmembrane receptors.     

Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ)

The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer.