RESUMO
G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and ß-arrestins. To influence specific GPCR signaling behaviors, ß-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within ß-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess ß-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.
Assuntos
Arrestinas , Receptores Acoplados a Proteínas G , beta-Arrestinas/metabolismo , Arrestinas/química , Arrestinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.
Assuntos
Anticorpos/imunologia , Western Blotting/métodos , Quinases de Receptores Acoplados a Proteína G/análise , Quinases de Receptores Acoplados a Proteína G/metabolismo , Animais , Células CHO , Cricetulus , Quinases de Receptores Acoplados a Proteína G/imunologia , Células HEK293 , Humanos , Isoenzimas , Camundongos , Células NIH 3T3 , Fosforilação , Ratos , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.
Assuntos
Quinases de Receptores Acoplados a Proteína G , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Humanos , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Quinases de Receptores Acoplados a Proteína G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Fosforilação , Animais , Transdução de SinaisRESUMO
ß-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-ß-arrestin complex. However, whether ß-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and ß-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of ß-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess ß-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.