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Integrazione temporale del segnale mediato da recettori accoppiati a proteine G: il potenziale ruolo di G15 nella cancerogenesi
Full text linkG15 è una proteina G eterotrimerica della famiglia di Gq/11, specifica del sistema ematopoietico, la cui funzione è sostanzialmente ignota. In questo studio si descrive la sua eccezionale resistenza al generale processo di desensibilizzazione che virtualmente regola l’attività di ogni recettore accoppiato a proteine G (GPCR).
Il segnale di G15 è infatti risultato refrattario alla desensibilizzazione del recettore β2 adrenergico indotta da sovraespressione di arrestina. Allo stesso modo, il livello d’espressione di arrestina non ha influito sul segnale mediato da G15 in risposta all’attivazione dei recettori V2 per la vasopressina e δ oppioide.
Esperimenti di co-immunoprecipitazione hanno dimostrato che la subunità Gα15 dell’eterotrimero (a differenza di Gαq and Gαs) viene stabilmente reclutata da un recettore V2 che una mutazione rende costitutivamente desensibilizzato. L’arrestina oltre a rendere meno efficiente il signalling dei GPCR, ne favorisce l’internalizzazione in compartimenti endosomiali. La co-espressione di Gα15 ha parzialmente ristabilito la presenza sulla membrana plasmatica del mutante costitutivamente desensibilizzato ristabilendone anche parte delle capacità segnalatorie. Tuttavia, confrontando l’effetto dell’agonista sull’attività verso gli effettori di G15, quali PKD1 e PLC, con l’effetto sull’internalizzazione del recettore, sono state osservate alcune peculiarità funzionali che suggeriscono che G15 stabilizzi una struttura conformazionale intermedia al normale processo di attivazione.
Nel loro insieme, questi risultati suggeriscono un nuovo meccanismo per cui la regolazione dei GPCR può essere aggirata e G15 può trasdurre segnali estremamente prolungati quando le cellule siano “cronicamente” esposte all’effetto di ormoni, neurotrasmettitori etc.
Questo aspetto ci ha portato ad ipotizzare che la presenza di G15 possa essere associata a stati fisiologici caratterizzati da fenomeni di deregolazione, in particolare con la formazione di tumori. Uno screening ha dimostrato la presenza di G15 in 10 fra 40 linee cellulari tumorali analizzate, tutte derivate da tessuti che in origine risultano negativi. Il messaggero di Gα15 e la corrispondente proteina sono anche risultati presenti in biopsie di cancro prostatico e pancreatico. Particolarmente rilevante appare il fatto che Gα15 fosse presente nella frazione proliferante del tumore così come dimostrato con xenotrapianti di tumore in topi nudi.G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization.
Enhancing β2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and δ opioid receptors.
Furthermore, co-immunoprecipitation experiments showed that G15 α subunit (as opposed to Gαq and Gαs) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by β-arrestin. Interestingly, co-expression of G15 partially rescued cell surface localization and signalling capabilities of the same mutant and supported its sustained activity on downstream effectors including PKD1. However, differential modulation by the agonist was observed while comparing effectors activity to receptor internalization.
Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters. This aspect led us to hypothesize that G15 presence could be associated to poorly regulated physiological states, and in particular with tumor formation. A screening demonstrated Gα15 presence in 10 out of 40 human cancer cell lines analyzed, all derived from a variety of tissues reported as negative.
Gα15 mRNA and protein were also found in pancreatic and prostate tumor biopsies. Notably, Gα15 was present in the proliferating fraction of the tumor as demonstrated by xenotransplantation of the tumors in nude mice
G protein-coupled receptor oligomerization provides the framework for signal discrimination.
No full textThe idea that G protein-coupled receptors (GPCRs) may undergo homo- or hetero-oligomerization, although highly controversial up to a few years ago, has recently gained wide acceptance. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based upon a large body of biochemical and biophysical evidence. While much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains rather elusive. GPCR oligomerization has been proposed to play a role in receptor ontogeny by either chaperoning protein folding or controlling trafficking to the cell surface. However, the acquisition of these roles does not rule out the possibility that oligomeric receptors may have additional functions, once they are brought to the cell surface. Herein, we propose that protein-protein as well as protein-lipid interactions may provide the structural basis for organizing distinct cell compartments along the plasma membrane where different extracellular signals may be perceived and discriminated
Nanoparticle-Based Oral Insulin Delivery: Challenges, Advances, and Future Directions
No full textExogenous insulin is essential for diabetes management; however, subcutaneous administration is associated with discomfort, poor adherence and non-physiological peripheral hyperinsulinemia. Oral administration would better mimic the physiological insulin distribution route but is hampered by gastrointestinal barriers, resulting in low bioavailability. Enabling access to the market for oral insulin nanocarriers requires rigorous control of their physicochemical attributes (size, charge, and surface chemistry) to ensure biocompatibility and mitigate risks such as the long-term bioaccumulation of non-biodegradable materials and the loss of intended targeting due to protein corona formation. In pre-clinical studies, nanoparticle carriers have shown promising results by protecting insulin and enhancing its absorption, yet clinical translation remains limited, with most candidates stalling in early-phase trials. This translational gap stems from the inadequacy of conventional animal models and regulatory frameworks to address the complexity of nanomedicines. This review goes beyond a simple summary of nanocarrier types and discusses the non-clinical and regulatory challenges hampering progress. We highlight the limitations of current preclinical models and the challenge of evaluating the pharmacokinetic profiles of both the nanocarrier and its insulin payload. The development of more rigorous and predictive strategies based on most recent successes and failures, described in this review, could help to bridge the translational gap
Glycosylation of V2 vasopressin receptor (V2R) assessed by metabolic labeling with S-35-methionine
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