The discovery of the new mechanism: Celastrol improves spinal cord injury by increasing cAMP through VIP-ADCYAP1R1-GNAS pathway.

Spinal cord injury (SCI) is a debilitating condition that results in significant impairment of motor function and sensation. Despite the ongoing efforts to develop effective treatments, there are currently very limited options available for patients with SCI. Celastrol, a natural anti-inflammatory compound extracted from Tripterygium wilfordii, has been shown to exhibit anti-inflammatory and anti-apoptotic properties. In this study, we aimed to explore the therapeutic potential of celastrol for SCI and elucidate the underlying molecular mechanisms involved. We found that local tissue often experiences a significant decrease in cAMP content and occurrs apoptosis after SCI. However, the treatment of celastrol could promote the production of cAMP by up-regulating the VIP-ADCYAP1R1-GNAS pathway. This could effectively inhibit the phosphorylation of JNK and prevent apoptosis, ultimately improving the exercise ability after SCI. Together, our results reveal celastrol may be a promising therapeutic agent for the treatment of SCI.

Allosteric modulation of adenosine A1 and cannabinoid 1 receptor signaling by G-peptides.

While allosteric modulation of GPCR signaling has gained prominence to address the need for receptor specificity, efforts have mainly focused on allosteric sites adjacent to the orthosteric ligand-binding pocket and lipophilic molecules that target transmembrane helices. In this study, we examined the allosteric influence of native peptides derived from the C-terminus of the Gα subunit (G-peptides) on signaling from two Gi-coupled receptors, adenosine A1 receptor (A1 R) and cannabinoid receptor 1 (CB1 ). We expressed A1 R and CB1 fusions with G-peptides derived from Gαs, Gαi, and Gαq in HEK 293 cells using systematic protein affinity strength modulation (SPASM) and monitored the impact on downstream signaling in the cell compared to a construct lacking G-peptides. We used agonists N6 -Cyclopentyladenosine (CPA) and 5'-N-Ethylcarboxamidoadenosine (NECA) for A1 R and 2-Arachidonoylglycerol (2-AG) and WIN 55,212-2 mesylate (WN) for CB1 . G-peptides derived from Gαi and Gαq enhance agonist-dependent cAMP inhibition, demonstrating their effect as positive allosteric modulators of Gi-coupled signaling. In contrast, both G-peptides suppress agonist-dependent IP1 levels suggesting that they differentially function as negative allosteric modulators of Gq-coupled signaling. Taken together with our previous studies on Gs-coupled receptors, this study provides an extended model for the allosteric effects of G-peptides on GPCR signaling, and highlights their potential as probe molecules to enhance receptor specificity.

Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the ß2-adrenergic receptor (ß2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the ß2AR stabilizes a 'closed' receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR­G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

Functional analysis of the interface regions involved in interactions between the central cytoplasmic loop and the C-terminal tail of adenylyl cyclase.

The mammalian adenylyl cyclase is a membrane-bound enzyme that is predicted to have 12 trans-membrane spans. Between membrane spans 6 and 7 there is a large cytoplasmic loop, which, along with the C-terminal tail, makes up the catalytic site of the enzyme. Crystal structures of these soluble cytoplasmic domains have identified the regions that are involved in interactions with each other. The functional consequences of these interactions in the full-length membrane-embedded enzymes have not been established. In this study, we analyzed the role of various interaction regions within the central cytoplasmic loop (C1) and the C-terminal tail (C2) on basal, Galphas-, forskolin-, and Mn(2+)-stimulated activities of adenylyl cyclases 2 and 6 (AC2 and AC6). We tested synthetic peptides encoding the different interface surfaces of both the C1 and C2 domain on different activities of membrane-bound AC2 and AC6 expressed in insect cells. We found the C1-alpha2-beta2-beta3 and C2-beta2'-beta3' regions to be involved in stimulation by Galphas and forskolin but not in the basal or Mn(2+)-stimulated activities. Both the C1-beta4-beta5-alpha4 region and the C2-alpha3'-beta4' region play a role in the Galphas- and forskolin-stimulated activities as well as in basal activity, because the peptides encoding these regions inhibit basal activity by 30%. In contrast, the C2-alpha2' region peptide inhibits both basal and Mn(2+)-stimulated activity by >50%. These results suggest that the different stimulated activities may involve distinct interface interactions in the intact enzyme and, consequently, the distinct mechanisms by which Mn(2+) activates the enzyme as compared with Galphas and forskolin, leading to the possibility that the full-length adenylyl cyclase may have multiple catalytically competent configurations.

Type VIII adenylyl cyclase in rat beta cells: coincidence signal detector/generator for glucose and GLP-1.

AIMS/HYPOTHESIS: The secretory function of pancreatic beta cells is synergistically stimulated by two signalling pathways which mediate the effects of nutrients and hormones such as glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) or glucagon. These hormones are known to activate adenylyl cyclase in beta cells. We examined the type of adenylyl cyclase that is associated with this synergistic interaction. METHODS: Insulin release, cAMP production, adenylyl cyclase activity, mRNA and protein expression were measured in fluorescence-activated cell sorter-purified rat beta cells and in the rat beta-cell lines RINm5F, INS-1 832/13 and INS-1 832/2. RESULTS: In primary beta cells, glucagon and GLP-1 synergistically potentiate the stimulatory effect of 20 mmol/l glucose on insulin release and cAMP production. Both effects are abrogated in the presence of the L-type Ca(2+)-channel blocker verapamil. The cAMP-producing activity of adenylyl cyclase in membranes from RINm5F cells is synergistically increased by Ca(2+)-calmodulin and recombinant GTP(gamma)S-activated G(s alpha)-protein subunits. This type of regulation is characteristic for type I and type VIII AC isoforms. Consistent with this functional data, AC mRNA analysis shows abundant expression of type VI AC, four splice variants of type VIII AC and low expression level of type I AC in beta cells. Type VIII AC expression at the protein level was observed using immunoblots of RINm5F cell extracts. CONCLUSION/INTERPRETATION: This study identifies type VIII AC in insulin-secreting cells as one of the potential molecular targets for synergism between GLP-1 receptor mediated and glucose-mediated signalling.

Activation of adenylyl cyclases, regulation of insulin status, and cell survival by G(alpha)olf in pancreatic beta-cells.

Because we recently identified the G(alpha)olf subunit in rat pancreatic beta-cells, we investigated the downstream effectors and the biological functions of this G protein in HEK-293T cells and the insulin-secreting mouse betaTC-3 cell line. With the use of transient transfection of HEK-293T cells with constitutively activated G(alpha)olf (G(alpha)olfQ214L, i.e., AG(alpha)olf), together with expression vectors encoding the adenylyl cyclase (AC) isoforms (AC-I to -VIII and soluble AC), compared with cotransfections using AG(alphas) (G(alphas)R201C), we observed that AG(alpha)olf preferentially activates AC-I and -VIII, which are also expressed in beta-cells. Stable overexpression of wild-type or AG(alpha)olf in betaTC-3 cells resulted in partial attenuation of insulin secretion and biosynthesis, suggesting that chronic activation of the G(alpha)olf-signaling pathway is associated with beta-cell desensitization. In agreement, transfected betaTC-3 cells present a decreased insulin content with respect to parental cells, whereas the proinsulin convertases PC-1 and PC-2 were unaffected. Furthermore, betaTC-3-AG(alpha)olf cells are resistant to serum starvation-induced apoptosis. Our findings suggest that G(alpha)olf is involved in insulin status, cell survival, and regeneration of the insulin-secreting beta-cells during development and diabetes.

Investigation of the role of the carboxyl-terminal tails of the alpha and beta isoforms of the human thromboxane A(2) receptor (TP) in mediating receptor:effector coupling.

We have investigated the functional coupling of alpha and beta isoforms of the human thromboxane A(2) receptor (TP) to Galpha(16) and Galpha(12) members of the G(q) and G(12) families of heterotrimeric G proteins in human embryonic kidney (HEK) 293 cell lines HEK.alpha10 or HEK.beta3, stably over-expressing TPalpha and TPbeta, respectively. Moreover, using HEK.TP(Delta328) cells which over-express a variant of TP truncated at the point of divergence of TPalpha and TPbeta, we investigated the requirement of the C-tail per se in mediating G protein coupling and effector activation. Both TPalpha and TPbeta couple similarly to Galpha(16) to affect increases in inositol 1,4,5-trisphosphate (IP(3)) and mobilisation of intracellular calcium ([Ca(2+)](i)) in response to the TP agonist U46619. Whilst both TP isoforms mediated [Ca(2+)](i) mobilisation in cells co-transfected with Galpha(12), neither receptor generated corresponding increases in IP(3), indicating that the Galpha(12)-mediated increases in [Ca(2+)](i) do not involve PLC activation. Verapamil, an inhibitor of voltage dependent Ca(2+) channels, reduced [Ca(2+)](i) mobilisation in TPalpha and TPbeta cells co-transfected with Galpha(12) to approximately 40% of that mobilised in its absence, whereas [8-(N,N-diethylamino)-octyl-3,4, 5-trimethoxybenzoate, hydrochloride] (TMB-8), an antagonist of intracellular Ca(2+) release, had no effect on [Ca(2+)](i) mobilisation by either receptor isoform co-transfected with Galpha(12). Despite the lack of differential coupling specificity by TPalpha and TPbeta, TP(Delta328) signalled more efficiently in the absence of a co-transfected G protein compared to the wild type receptors but, on the other hand, displayed an impaired ability to couple to co-transfected Galpha(11), Galpha(12) or Galpha(16) subunits. In studies investigating the role of the C-tail in influencing coupling to the effector adenylyl cyclase, similar to TPalpha but not TPbeta, TP(Delta328) coupled to Galpha(s), leading to increased adenosine 3',5'-cyclic monophosphate (cAMP), rather than to Galpha(i). Whereas TP(Delta328) signalled more efficiently in the absence of co-transfected G protein compared to the wild type TPalpha, co-transfection of Galpha(s) did not augment cAMP generation by TP(Delta328). Hence, from these studies involving the wild type TPalpha, TPbeta and TP(Delta328), we conclude that the C-tail sequences of TP are not a major determinant of G protein coupling specificity to Galpha(11) and Galpha(16) members of the G(q) family or to Galpha(12); it may play a role in determining G(s) versus G(i) coupling and may act as a determinant of coupling efficiency.

Analysis of sequence-dependent interactions between transient calcium and transmitter stimuli in activating adenylyl cyclase in Aplysia: possible contribution to CS--US sequence requirement during conditioning.

An important recent insight in a number of neurobiological systems is that during learning, individual dually regulated proteins with associative properties function as critical sites of stimulus convergence. During conditioning in Aplysia, the Ca2+ /calmodulin-sensitive adenylyl cyclase (AC) in mechanosensory neurons serves as a molecular site of interaction between Ca2+ and serotonin [5-hydroxytryptamine (5-HT)]-two signals that represent the CS and US in these cells. Conditioning requires that the CS and US be paired within a narrow time window and in the appropriate sequence. AC shows an analogous sequence preference: It is more effectively activated when a pulse of Ca2+ precedes a pulse of 5-HT than when the 5-HT precedes Ca2+. One mechanism that contributes to this sequence preference is that Ca2+/calmodulin binding to AC accelerates the rate of AC activation by receptor-Gs. We have identified two additional properties of AC activation that would cause pairing with Ca2+ preceding 5-HT to be more effective than simultaneous pairing or pairing with the reciprocal sequence: (1) Activation of Aplysia AC by a Ca2+ pulse rose with a delay compared with activation by a 5-HT pulse. (2) A late pulse of Ca2+, which arrived after 5-HT, acted, via calmodulin, to accelerate the decay of AC activation by receptor-Gs. Together, these activation properties of AC may contribute to the CS-US sequence requirement of classical conditioning.