Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1–Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1–Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.Cells detect and respond to a myriad of extracellular signals via G-protein signaling pathways. G proteins form complexes consisting of Gα, Gβ, and Gγ subunits that play a key role in propagating signals between activated receptors and downstream effectors (1). To perform this role, the G-protein heterotrimer must be assembled from its nascent polypeptides. A critical step in this process is the formation of the Gβγ dimer (2). Gβγ is an obligate dimer in which the individual subunits cannot fold into a stable structure on their own, but require the molecular chaperone cytosolic chaperonin containing tailless complex polypeptide 1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1) (3, 4).CCT is a member of the group II chaperonin family found in eukaryotes. It is a large protein-folding machine, made up of eight homologous subunits that assemble to form a double-ring structure, with each ring encompassing a central cavity. Nascent polypeptides and denatured proteins bind these cavities and are thereby sequestered from the other proteins in the cytosol and protected from aggregation (5). Each of the eight CCT subunits is an ATPase, and the binding and hydrolysis of ATP generates a conformational change in CCT that encapsulates the protein and assists in its folding (6–8). An important class of CCT substrates is WD40 repeat proteins that form β-propeller structures (9). One of these WD40 substrates is Gβ, which forms a seven-bladed β-propeller (10) with the assistance of CCT (3). However, unlike other CCT substrates, Gβ cannot achieve its native structure and release from CCT on its own, but requires the help of PhLP1 (3, 4). PhLP1 triggers the release of Gβ from CCT, allowing Gβ to interact with Gγ and form the Gβγ dimer (3, 4, 11).Given their vital roles in Gβγ assembly, it is important to understand at the molecular level how CCT and PhLP1 mediate Gβ folding. To achieve this objective, we have isolated two key intermediates in the Gβγ assembly process, the Gβ-CCT complex and the PhLP1–Gβ-CCT complex, and examined their structures by cryo-electron microscopy (cryo-EM), site-specific chemical cross-linking using unnatural amino acids, and chemical cross-linking coupled with mass-spectrometric identification of the cross-links (XL-MS). This analysis reveals a complex molecular mechanism by which CCT and PhLP1 fold Gβ and assist in Gβγ assembly.     

Development of the mammalian axial skeleton requires signaling through the Gαi subfamily of heterotrimeric G proteins

129/SvEv mice with a loss-of-function mutation in the heterotrimeric G protein α-subunit gene Gnai3 have fusions of ribs and lumbar vertebrae, indicating a requirement for Gα_i (the “inhibitory” class of α-subunits) in somite derivatives. Mice with mutations of Gnai1 or Gnai2 have neither defect, but loss of both Gnai3 and one of the other two genes increases the number and severity of rib fusions without affecting the lumbar fusions. No myotome defects are observed in Gnai3/Gnai1 double-mutant embryos, and crosses with a conditional allele of Gnai2 indicate that Gα_i is specifically required in cartilage precursors. Penetrance and expressivity of the rib fusion phenotype is altered in mice with a mixed C57BL/6 × 129/SvEv genetic background. These phenotypes reveal a previously unknown role for G protein-coupled signaling pathways in development of the axial skeleton.     

Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700

L-type calcium (Ca²⁺) currents conducted by voltage-gated Ca²⁺ channel Ca_V1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca_V1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca²⁺ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the Ca_V1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca_V1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca²⁺ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.Upon membrane depolarization, Ca_V1.2 channels conduct L-type calcium (Ca²⁺) current into cardiomyocytes and initiate excitation–contraction coupling (1, 2). Ca²⁺ influx through Cav1.2 channels activates Ca²⁺ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation–contraction coupling, Ca²⁺ influx via Ca_V1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca_V1.2 channel (1, 3). Phosphorylation of the Ca_V1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca_V1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4–6). Under those pathological conditions, responsiveness of Ca_V1.2 channel activity to β-adrenergic receptors and PKA activation is severely blunted, resulting in diminished contractile reserve and impaired fight-or-flight response (6, 7). Enormous effort has been devoted to understanding how β-adrenergic regulation of the Ca_V1.2 channel is achieved, but the exact molecular mechanisms remain unresolved.Ca_V1.2 channels contain multiple subunits, including a pore-forming α₁1.2 subunit (also designated α_1C), β and α₂δ subunits that modulate expression of Ca_V1.2 at the cell surface, and possibly γ subunits (8). The closely related Ca_V1.1 and Ca_V1.2 channels in skeletal and cardiac muscle, respectively, are both proteolytically processed near the center of their large C-terminal domains (9, 10), and the distal C terminus (dCT) remains associated noncovalently with the proximal C terminus (pCT) and serves as a potent autoinhibitor (11, 12). Regulation of Ca_V1.2 channels by PKA was reconstituted in nonmuscle cells with a dynamic range of threefold to fourfold similar to native cardiomyocytes by building the autoinhibitory Ca_V1.2 complex through cotransfection of each of its components (13). Successful reconstitution required an A Kinase Anchoring Protein (AKAP), which recruits PKA to the dCT (13–15). Deletion of the dCT in vivo results in loss of regulation of the L-type Ca²⁺ current by the β-adrenergic pathway and embryonic death from heart failure (16, 17). These results suggest that the autoinhibited Ca_V1.2 signaling complex serves as the substrate for β-adrenergic regulation, and disruption of this complex leads to heart failure.PKA is responsible for phosphorylation of the Ca_V1.2 channel in response to β-adrenergic stimulation in cardiac myocytes (18–22). Although multiple PKA sites have been identified in α₁ subunits by in vitro phosphorylation (10, 23), none of these sites is required for regulation of Ca_V1.2 channels in vivo. For example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10, 24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca_V1.2 channel activity in transfected cells or cardiomyocytes (13, 25, 26). Two sites in the C terminus of the skeletal muscle Ca_V1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27). These sites are conserved in cardiac Ca_V1.2 channels as S1700 and T1704, and phosphoproteomics analysis revealed β-adrenergic–stimulated phosphorylation of S1700 by PKA (28). S1700 and T1704 reside at the interface between the pCT and dCT. In studies of the Ca_V1.2 signaling complex reconstituted in nonmuscle cells, phosphorylation of both sites was required for normal basal channel activity, whereas only S1700 was essential for PKA stimulation (13). Mutation of S1700 and T1704 to Ala in STAA mice reduced basal activity and Ca_V1.2 channel regulation by the β-adrenergic pathway in cardiomyocytes (29). To further dissect the contribution of S1700, we studied a mutant mouse line expressing Ca_V1.2 channel with the S1700A mutation (SA mice). Our results demonstrate that this single phosphorylation site is required for normal regulation of Ca_V1.2 channels, contraction of cardiac myocytes, exercise capacity, and cardiac homeostasis.     

Taking the heart failure battle inside the cell: small molecule targeting of Gβγ subunits

Heart failure (HF) is devastating disease with poor prognosis. Elevated sympathetic nervous system activity and outflow, leading to pathologic attenuation and desensitization of β-adrenergic receptors (β-ARs) signaling and responsiveness, are salient characteristic of HF progression. These pathologic effects on β-AR signaling and HF progression occur in part due to Gβγ-mediated signaling, including recruitment of receptor desensitizing kinases such as G-protein coupled receptor (GPCR) kinase 2 (GRK2) and phosphoinositide 3-kinase (PI3K), which subsequently phosphorylate agonistoccupied GPCRs. Additionally, chronic GPCR signaling signals chronically dissociated Gβγ subunits to interact with multiple effector molecules that activate various signaling cascades involved in HF pathophysiology. Importantly, targeting Gβγ signaling with large peptide inhibitors has proven a promising therapeutic paradigm in the treatment of HF. We recently described an approach to identify small molecule Gβγ inhibitors that selectively block particular Gβγ functions by specifically targeting a Gβγ protein-protein interaction "hot spot." Here we describe their effects on Gβγ downstream signaling pathways, including their role in HF pathophysiology. We suggest a promising therapeutic role for small molecule inhibition of pathologic Gβγ signaling in the treatment of HF. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure.”     

The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ₁γ₂ dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gα_s and Gα_i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ₁ orthologs, Gβ₁ and Gβ_1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ₁/Gβ_1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.     

Modulation of voltage-gated K+ channels by the sodium channel ��1 subunit

Voltage-gated sodium (Na_V) and potassium (K_V) channels are critical components of neuronal action potential generation and propagation. Here, we report that Na_Vβ1 encoded by SCN1b, an integral subunit of Na_V channels, coassembles with and modulates the biophysical properties of K_V1 and K_V7 channels, but not K_V3 channels, in an isoform-specific manner. Distinct domains of Na_Vβ1 are involved in modulation of the different K_V channels. Studies with channel chimeras demonstrate that Na_Vβ1-mediated changes in activation kinetics and voltage dependence of activation require interaction of Na_Vβ1 with the channel’s voltage-sensing domain, whereas changes in inactivation and deactivation require interaction with the channel’s pore domain. A molecular model based on docking studies shows Na_Vβ1 lying in the crevice between the voltage-sensing and pore domains of K_V channels, making significant contacts with the S1 and S5 segments. Cross-modulation of Na_V and K_V channels by Na_Vβ1 may promote diversity and flexibility in the overall control of cellular excitability and signaling.     

Structural elements in the Girk1 subunit that potentiate G protein–gated potassium channel activity

G protein–gated inwardly rectifying K⁺ (Girk/K_IR3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABA_B receptor and Girk1/Girk2 heteromer as a model system. Girk1 did not increase the protein levels or alter the trafficking of Girk2-containing channels to the cell surface in transfected cells or hippocampal neurons, indicating that its potentiating influence involves enhancement of channel activity. Structural elements in both the distal carboxyl-terminal domain and channel core were identified as key determinants of robust channel activity. In the distal carboxyl-terminal domain, residue Q404 was identified as a key determinant of receptor-induced channel activity. In the Girk1 core, three unique residues in the pore (P) loop (F137, A142, Y150) were identified as a collective potentiating influence on both receptor-dependent and receptor-independent channel activity, exerting their influence, at least in part, by enhancing mean open time and single-channel conductance. Interestingly, the potentiating influence of the Girk1 P-loop is tempered by residue F162 in the second membrane-spanning domain. Thus, discontinuous and sometime opposing elements in Girk1 underlie the Girk1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity.     

Rapid β-lactam-induced lysis requires successful assembly of the cell division machinery

β-lactam antibiotics inhibit penicillin binding proteins (PBPs) involved in peptidoglycan synthesis. Although inhibition of peptidoglycan biosynthesis is generally thought to induce cell lysis, the pattern and mechanism of cell lysis can vary substantially. β-lactams that inhibit FtsI, the only division specific PBP, block cell division and result in growth as filaments. These filaments ultimately lyse through a poorly understood mechanism. Here we find that one such β-lactam, cephalexin, can, under certain conditions, lead instead to rapid lysis at nascent division sites through a process that requires the complete and ordered assembly of the divisome, the essential machinery involved in cell division. We propose that this assembly process (in which the localization of cell wall hydrolases depends on properly targeted FtsN, which in turn depends on the presence of FtsI) ensures that the biosynthetic machinery to form new septa is in place before the machinery to degrade septated daughter cells is enabled. β-lactams that target FtsI subvert this mechanism by inhibiting FtsI without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. One seemingly paradoxical implication of our results is that β-lactam therapy may be improved by promoting active cell division.     

Expression of mitochondrial fusion–fission proteins during post-infarction remodeling: the effect of NHE-1 inhibition

Studies on the role of mitochondrial fission/fusion (MFF) proteins in the heart have been initiated recently due to their biological significance in cell metabolism. We hypothesized that the expression of MFF proteins is affected by post-infarction remodeling and in vitro cardiomyocyte hypertrophy, and serves as a target for the Na⁺/H⁺ exchanger 1 (NHE-1) inhibition. Post-infarction remodeling was induced in Sprague–Dawley rats by coronary artery ligation (CAL) while in vitro hypertrophy was induced in cardiomyocytes by phenylephrine (PE). Mitochondrial fission (Fis1, DRP1) and fusion (Mfn2, OPA1) proteins were analyzed in heart homogenates and cell lysates by Western blotting. Our results showed that 12 and 18 weeks after CAL, Fis1 increased by 80% (P < 0.01) and 31% (P < 0.05), and Mfn2 was reduced by 17% (P < 0.05) and 22% (P < 0.05), respectively. OPA1 was not changed at 12 weeks, although its expression decreased by 18% (P < 0.05) with 18 weeks of ligation. MFF proteins were also affected by PE-induced hypertrophy that was dependent on mitochondrial permeability transition pore opening and oxidative stress. The NHE-1-specific inhibitor EMD-87580 (EMD) attenuated changes in the expression of MFF proteins in both the models of hypertrophy. The effect of EMD was likely mediated, at least in part, through its direct action on mitochondria since Percoll-purified mitochondria and mitoplasts have been shown to contain NHE-1. Our study provides the first evidence linking cardiac hypertrophy with MFF proteins expression that was affected by NHE-1 inhibition, thus suggesting that MFF proteins might be a target for pharmacotherapy with anti-hypertrophic drugs.     

Reception of Slit requires only the chondroitin–sulphate-modified extracellular domain of Syndecan at the target cell surface

Syndecan (Sdc) is a conserved transmembrane heparan sulfate proteoglycan (HSPG) bearing additional chondroitin sulfate (CS) modifications on its extracellular domain. In vertebrates, this extracellular domain of Sdc is shed and acts as a soluble effector of cellular communication events, and its cytoplasmic domain participates in intracellular signaling needed to maintain epithelial integrity. In Drosophila, Sdc has been shown to be necessary for Slit signaling-dependent axon and myotube guidance during CNS development and muscle pattern formation. We report that Sdc acts in a cell-autonomous manner in Slit-receiving cells and that its membrane-anchored extracellular domain is sufficient to mediate Slit signaling. Sdc activity can be replaced by the human homolog hsdc2. However, the HSPG Dally-like protein (Dlp), which lacks CS modifications at its extracellular domain, can only partially substitute for Sdc function, and its activity is not restricted to the Slit target cells. Our results suggest that Sdc and Dlp act in a cooperative but nonredundant fashion in axon and myotube guidance. We propose that Dlp, which lacks CS modifications, participates in the transfer of Slit from its site of expression to the target cells, where CS-modified Sdc concentrates and presents the ligand.     

What is the role of renin inhibition in the treatment of diabetic kidney disease?

Inhibition of the renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in preventing and treating diabetic nephropathy. However, despite documented beneficial effects of RAAS inhibitors in diabetic patients with nephropathy, reversal of the progressive course of this disorder or at least long-term stabilization of renal function are often difficult to achieve, and many patients still progress to end-stage renal disease. Incomplete inhibition of the RAAS has been postulated as one of the reasons for unsatisfactory therapeutic responses to RAAS inhibition in some patients. Inhibition of renin, a rate-limiting step in the RAAS activation cascade, is the logical approach to overcome at least some of the above-mentioned problems associated with the treatment with traditional RAAS inhibitors. This article focuses on experimental and clinical studies evaluating the two principal approaches to renin inhibition: direct renin inhibition with competitive inhibitors (eg, aliskiren) and inhibition of the (pro)renin receptor.