Browsing by Subject "beta-arrestin"

Sonic hedgehog (Shh) is a critical secreted signaling molecule that regulates many aspects of organogenesis. In the absence of Shh, many organs, including the foregut, larynx, palate, cerebellum and heart do not form properly. However, the cellular details of the roles of Shh, including the relevant domains of Shh expression and reception, have not been elucidated for many of these processes.

The single embryonic foregut tube must divide into the trachea and esophagus, which does not occur in the Shh-null mutant. In Chapter 5, I use Cre-Lox technology to determine that the ventral foregut endoderm is the relevant source of Shh for this process and the mesoderm must directly receive that Shh signal. Surprisingly, this signaling event appears to occur two days before the foregut begins to divide, indicating an early essential role for Shh in foregut division.

Shh is also expressed at later stages in the maturing trachea and esophagus. In Chapter 6, I demonstrate that these domains serve to establish differentiated mesoderm. In the trachea, Shh from the endoderm signals directly to the mesoderm to form the tracheal cartilage rings. In the esophagus, the roles of Shh are more complex. Shh regulates the size of the esophagus and controls patterning of the concentric rings of esophageal mesoderm, however this process seems to be indirect, requiring autocrine Shh signaling within the esophageal endoderm.

The laryngeal apparatus is entirely absent in the Shh-null mouse. I n Chapter 3, I dissect the domains of Shh expression and reception required for laryngeal development and demonstrate that loss of endodermal Shh expression causes laryngotracheoesophageal clefts and malformed laryngeal cartilages. As much of laryngeal morphogenesis poorly understood, I also utilize dual mesodermal and neural crest fate maps to determine the embryonic origins of various laryngeal tissues. Finally, as Shh signaling often occurs in concert with Bone Morphogenic Protein (BMP) signaling, I investigate the roles of BMP signaling in laryngeal development.

Much of Shh signaling occurs at the primary cilium, to which Smoothened, a critical pathway member, must translocate upon Shh signal transduction. This process requires a Smo-Kif3a-βarretin complex in mammalian cell culture. However, the roles of βarrestins in mouse development, and their relationship to Shh signaling have not been investigated in vivo. To do so, in Chapter 4, I analyze the phenotypes of the βarr1/βarr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling.

Altogether, my work dissects the cellular details of Shh signaling during multiple organ systems in the mouse embryo. I further analyze the consequences of absent or misregulated Shh signaling across multiple developmental contexts and determine that Shh plays critical and diverse roles in organogenesis.

G protein-coupled receptors (GPCRs) represent the largest and the most versatile family of cell surface receptors. Members of this receptor family translate diverse extracellular cues to intracellular responses, and are commonly targeted for medicinal therapeutics. In the current model of GPCR signaling, agonist binging not only initiates G protein-mediated signaling through generation of second messengers such as cyclic AMP and diacylglycerol, but also through the multifunctional adaptor protein beta-arrestin acting as a signal transducer. While some ligands have balanced agonist activity defined as equal efficacy for G protein and beta-arrestin-mediated pathways, other ligands stimulates GPCR signaling preferentially through beta-arrestin, a concept know as beta-arrestin-biased agonism.

The beta1 and beta2 adrenergic receptors (betaARs) are the predominant GPCR subtypes expressed in the heart, and play an important role in the pathophysiology of human heart disease. Of the four families of G alpha-proteins (G alpha s, G alpha i/o, G alpha q/11 and G alpha 12/13), beta1ARs are recognized as classical G alpha s-coupled receptors since agonist binding promotes coupling to heterotrimeric G protein (G alpha-beta-gamma) triggering dissociation of G alpha s from G beta-gamma. Here, we identify a new signaling mechanism unlike that previously for any known G alpha s-coupled receptor whereby the inhibitory G protein (G alpha i) is required for beta1AR-mediated beta-arrestin-biased signaling. Stimulation with the beta-arrestin-biased agonist carvedilol induces switching of the beta1AR from a classical G alpha s-coupled receptor to a G alpha i-coupled receptor and stabilizes a unique receptor conformation required for beta-arrestin-mediated signaling. Recruitment of G alpha i was not induced by any other betaAR agonist or antagonist screened, nor was it required for beta-arrestin-bias activated by the beta2AR subtype of the betaAR family.

We also found that G alpha i is involved in the membrane stretch-induced signaling activated by the angiotensin II type 1 receptor (AT1R), another GPCR mediating a variety of physiological responses and is commonly targeted for cardiac drug therapy. It is appreciated that the AT1R can function as a mechanical sensor. When activated by mechanical stretch, AT1Rs transmit signaling through beta-arrestin, rather than the G protein pathways. To date, the ligand-independent membrane stretch-induced AT1R conformation that triggers signaling is thought to be the same as that induced by a beta-arrestin-biased agonist, which can selectively engage beta-arrestin and prevent G protein coupling. Here we show that membrane stretch promotes a distinct biased conformation of the AT1R that couples to G alpha i. In contrast, recruitment of G alpha i was not induced by the balanced agonist angiotensin II, nor by the beta-arrestin-biased agonist TRV120023. The stretch-triggered G alpha i coupling induces the recruitment and a unique conformational change in beta-arrestin2 allowing for downstream signaling such as EGFR internalization and ERK phosphorylation.

Taken together, we identified a previously unrecognized role for G alpha i in beta-arrestin-biased GPCR signaling, and suggests that the concept of beta-arrestin-bias may need to be refined to incorporate the selective bias of receptors towards distinct G protein subtypes. We also demonstrate that different mechanisms for beta-arrestin bias may be operative between the signaling induced by distinct receptor activation modes, in the case of AT1R, the beta-arrestin-biased agonists and by the ligand-independent mechanical stretch.

These data underscore the complexity of beta-arrestin-biased agonism and have important implications when considering the development of new therapeutic ligands to selectively target beta-arrestin-biased signaling pathways.

Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby, in turn, instruct its functional capabilities.

We determined conformational changes in beta-arrestin1 in vitro using limited tryptic proteolysis and MALDI-TOF MS analysis in the presence of a phosphopeptides derived from the C-terminus of the V2 vasopressin receptor (V2Rpp or V2R4p) or the corresponding unphosphorylated peptide (V2Rnp). Upon V2Rpp binding, we show that the previously shielded R393 becomes accessible, which indicates release of the C-terminus. Moreover, we have shown that R285 becomes more accessible and this residue is located in a region of β-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1. We also show a functional consequence of the release of beta-arrestin1's C-terminus by enhanced clathrin binding. In addition, we have shown marked protection of beta-arrestin1's N-domain in the presence of V2Rpp; consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. Using a differentially phsophorylated V2R peptide (V2R4p), we show that beta-arrestin1 is able to adopt distinct conformations in response to different phosphorylation patterns. Futhermore, a striking difference is observed in the conformation of V2Rpp-bound beta-arrestin1 when compared to beta-arrestin2, namely the flexibility of the inter-domain hinge region. These data represent the first direct evidence that the beta-arrestin1 conformation is differentially instructed by phosphorylation patterns and that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.

Phosphorylation of 7TMRs by GRKs plays essential roles in regulation of receptor function by promoting interactions of the receptors with beta-arrestins. We hypothesized that different GRKs phosphorylate distinct sets of sites thereby establishing a "bar code." In order to test this hypothesis, we monitored the phosphorylation events of the beta2AR upon stimulation with a classical full agonist, isoproterenol, or a beta-arrestin "biased" agonist, carvedilol, in the presence of a full complement of GRKs or when individual GRKs (2 or 6) were depleted by siRNA. We demonstrate that at least thirteen sites on the beta2AR show changes in phosphorylation in response to the agonist isoproterenol. Of these, phosphorylation increased 10 to more than 300 fold in 12 (S261, S262, S345, S346, S355, S356, T360, S364, S396, S401, S407 AND S411) and decreased 50% in one (S246). Depletion of GRK2 or 6 by siRNA indicates that S355, 356 are GRK6 sites whereas the remainder are GRK2 sites. Phosphorylation of GRK2 sites inhibits that of GRK6 sites. Carvedilol, a beta-arrestin biased agonist, promotes phosphorylation of only the GRK6 sites S355, 356. In HEK293 cells, GRK2 phosphorylation is found to be the major positive regulator of receptor internalization; to contribute to receptor desensitization; and to inhibit beta-arrestin mediated ERK activation. Phosphorylation of the two GRK6 sites contributes to receptor desensitization and internalization and is required for beta-arrestin mediated ERK activation. These data indicate that different ligands promote distinct patterns of receptor phosphorylation which dictate different patterns of beta-arrestin mediated function.

Dopamine receptors (D1-5R), responsible for cognition and locomotion, are involved in several neurological diseases. Parkinson’s Disease (PD) is a neurodegenerative disorder wherein patients suffer from the loss of dopaminergic neurons, resulting in both bradykinesia and tremors. Levodopa (L-DOPA) and apomorphine are both effective treatments of PD; however, they both produce severe dyskinesia with chronic use. In a PD model, a recent discovery shows the G-protein pathway is associated with dyskinesia while the beta-arrestin pathway led to locomotor improvement. As of date, there are no known beta-arrestin biased ligands at D1R. Thus, this work focuses on the derivatization of (R)-apomorphine to develop a D1- or D2R beta-arrestin biased ligand through total synthesis or through late-stage functionalization of the core. These analogs will be tested in vitro through collaboration where we will study the structure-functional-selectivity relationship to better assist us in the design of future beta-arrestin biased analogs. The long-term goal of this research is to contribute to the improvement of PD treatment through selective activation of the beta-arrestin pathway. This work is motivated by the severity of L-DOPA induced dyskinesia and the lack of proper, long-term treatment.