Beta-Arrestin Mediated Signaling in the Heart

The classical mechanism of β-receptor activation is through G protein-dependent signaling of the 7 transmembrane receptors, the G protein coupling receptors (GPCR).  A key molecule is the G-protein coupled receptor kinase (GRK), which phosphorylates the 7 transmembrane receptor on the C-terminal tail of the molecule. After this phosphorylation, β-arrestin binds and terminates the signal. This termination is known as desensitization. 

However, this classic system is more complicated than initially understood. When the receptor is desensitized, it undergoes trafficking and signaling by different pathways. The β-receptors are abnormal in heart failure. In human heart failure, the β1 receptor is downregulated, about half of the receptors for the β1 receptor are decreased on the cell surface, and the β2 receptor is desensitized. An increase in the β-adrenergic receptor kinase (βARK) diminishes the signaling pathway that increases heart rate and contractility. Rockman and colleagues, and others, have been working to understand this desensitization process and why the receptor is downregulated, and if this is part of the mechanism in the failing heart.

Beta-adrenergic receptors and MI

Known β-adrenergic receptors (βAR) in the human heart are β1, β2, and β3. Whether the β1 and  β2 receptors are antagonistic is controversial. Some reports show that the β2 receptor activates protective pathways, primarily through phosphoinositide 3-kinase (PI3K). It is unclear if the β2 receptor is protective in the heart in vivo.

Rockman and colleagues showed that cardiac function was worse after myocardial infarction (MI) in the presence of β1 receptors in experimental knockout animals. β1AR promotes an increase in cMAP kinase activity post MI, equal to that seen if just catecholamines were given to these animals, which is a  β1AR subtype-specific signaling event.  No increase in cMAP kinase activity was seen in the double knockout mice or the β2 receptor only mice. Further, they showed an increase in apoptosis in the presence of only β1AR. Thus, β1AR is very important for the acute response to catecholamines, heart rate, contractility, but chronically β1AR appear to promote left ventricular dysfunction, increase cMAP kinase activity signaling under catecholamines and post-MI, and are associated with increased apoptosis in the border zone of an infarction. The β2AR do not seem to play a protective role, and the absence of the β1AR was the most important feature in the deterioration of the heart, not the presence of the β2AR, in this work by Rockman and colleagues. 

Receptor localized phosphoinositide 3-Kinase (PI3K) and βAR trafficking

PI3K is a central molecule in the internalization in trafficking of the βAR. Rockman reviewed a new concept of a very active signaling complex. Cytoplasmic GRK is translocated to the receptor where it phosphorylates on the C terminal portion and induces desensitization and recruits the PI3K molecule.  Work by Rockman and colleagues show that PI3K has two functions, protein kinase activity and lipid kinase activity. 

In the GRK complex, β-arrestin allows for binding of clathrin and a molecule of AP2 and generates phosphoinositide 3 phosphate (PIP3), allowing for internalization of the βAR via or through a clathrin-mediated vesicle. This endocytosis or internalization process recycles receptors and importantly activates signaling pathways. 

PI3K in the cell takes phosphatidyl inositol 4,5 phosphate and adds a phosphate to the lipositol ring to generate phosphatidyl inositol 3,4,5 phosphate (PIP3). Rockman and colleagues discovered that this process occurs in the membrane associated with the βAR.

The PIK domain binds PRK and goes to the membrane. The catalytic domain contains protein activity and lipid kinase activity. Rockman and colleagues showed in animal models that it is possible to block internalization of βAR trafficking, keeping PI3K on the cell surface. They also showed that both protein and lipid phosphorylation activity is required for trafficking and the βAR to enter the cell.

Prasad and Rockman showed that PI3K phosphorylates a molecule called cytoskeletal tropomyosin, which allows for internalization. Thus, PI3K generates PIP3 and these phospholipids promote β-arrestin  binding to the phosphorylated receptor and promotes the binding of actin and clathrin. PI3K also phosphorylates tropomyosin, so the clathrin-coated vesicle internalizes the actin and tropomyosin bridges that are formed.  

β-arrestin mediated EGFR transactivation

Important signaling pathways believed to be important in normal and abnormal hearts are activated when the βAR is internalized or there is trafficking in this complex. A new concept is β-arrestin mediated epidermal growth factor receptor (EGFR) transactivation.

Stimulated GPCR, such as the angiotensin receptor, can activate a metalloproteinase to cleave a an EGF-like ligand from the surface to stimulate EGFR receptors to enter the cell.  Work by Rockman and colleagues show the β1AR strongly transactivates the EGFR, a process that requires β-arrestin and phosphorylation of the receptor. Further, they showed this is an important intracellular signaling pathway in the heart. 

Experimental work showed that in response to catecholamines, dobutamine, and isoproterenol EGFR enters the cell, it is internalized and activated.  The EGF ligand also stimulates EGFR to enter the cell. Further, they were able to block transactivation of β1AR through EGFR if β-arrestin was eliminated.

In βAR mutant mice without phosphorylation sites, Rockman’s group showed that the wild type receptor transactivates EGFR by dobutamine stimulation, phosphorylation of the EGFR, or activation or internalization of the EGFR with catecholamines.  In contrast, there is no phosphorylation of the EGFR, very little ERK, and no internalization of the EGFR into the cell.  The wild type β1AR can transactivate EGFR, but GRK cannot.

Further work showed that GRK 5 and GRK6 are critical for transactivation. They also demonstrated the importance of transactivation compared to direct EGF stimulation. 

Transactivation of the EGFR receptor activates ERK, but ERK stays in the cytoplasm. In contrast, directly adding the EGF ligand causes tremendous ERK phosphorylation, but virtually all the ERK goes to the nucleus. In the presence of dobutamine, there is no increase in transcription through ERK-mediated events.  In contrast, in the presence of EGF, a 2-fold increase in transcriptional events directly related to ERK is seen; this is blocked by the EGFR. 

Evidence from a variety of studies show that a complex of two receptors (βAR and EGFR) is the mechanism by which ERK stays in the nucleus or stays in the cytoplasm. When the βAR is activated by catecholamine, it binds β-arrestin, transactivates the EFG and it is internalized, and ERK stays in that complex in the cytoplasmic cytosol.  In contrast, when the EGFR is activated directly by ligand, it does not use the βAR or β-arrestin.  Therefore, ERK is free to translocate to the nucleus directly.  This compartmentalization of ERK is a key response to the signaling pathways that are activated by either the β-receptor agonist or EGFR agonist. 

β1AR-stimulated EGFR transactivation in vivo

In transgenic animals (wild type β1AR, mutants of the β1AR known as GRK receptor, or mutant β1AR receptor known as the PKA receptor) they showed that the GRK receptor cannot activate myocardial ERK/Akt signaling in the GRK transgenic animal but is normal in the wild type or the PKA mice.  Other studies demonstrated the animals behave the same in vivo and in vitro, meaning that when the β1AR is stimulated it transactivates the EGF in the heart in the wild type animal but not in the GRK receptor animals.

The transgenic mice overexpressing the wild type β1AR before and after isoproterenol is normal but the GRK receptor mice is abnormal after seven days of isoproterenol.  Fractional shortening falls by 15-20% after only one week of isoproterenol stimulation.  An increase in apoptosis was found in association with the fall in fractional shortening in the GRK hearts, but not in the wild type, β1 hearts, normal hearts, or the PKA heart. In fact, nearly a 3-fold increase in cellular apoptosis was found in the GRK-heart compared to the other hearts. They believe this in part accounts for the deterioration of cardiac function. 

The in vitro and in vivo data show that β1AR transactivate the EGFR. This signal from transactivation is different than from ligand stimulation. The transactivation is a cardioprotective signal, because they showed that the heart deteriorates if the transactivation is lost.

βARK inhibition promotes transactivation of the epidermal growth factor. Prevention of GRK2 phosphorylation promoted GRK5 and GRK6 phosphorylation of the βAR and allows transactivation to proceed. Rockman and colleagues believe this is cardioprotective.