Cholesterol Dependent Activity of the Adenosine A2A Receptor Is Modulated via the Cholesterol Consensus Motif.

BACKGROUND: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. METHODS: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. RESULTS AND CONCLUSIONS: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.

Transient ammonia stress on Chinese hamster ovary (CHO) cells yield alterations to alanine metabolism and IgG glycosylation profiles.

BACKGROUND: Ammonia concentrations typically increase during mammalian cell cultures, mainly due to glutamine and other amino acid consumption. An early ammonia stress indicator is a metabolic shift with respect to alanine. To determine the underlying mechanisms of this metabolic shift, a Chinese hamster ovary (CHO) cell line with two distinct ages (standard and young) was cultured in parallel fed-batch bioreactors with 0 mM or 10 mM ammonia added at 12 h. Reduced viable cell densities were observed for the stressed cells, while viability was not significantly affected. The stressed cultures had higher alanine, lactate, and glutamate accumulation. Interestingly, the ammonia concentrations were similar by Day 8.5 for all cultures. We hypothesized the ammonia was converted to alanine as a coping mechanism. Interestingly, no significant differences were observed for metabolite profiles due to cell age. Glycosylation analysis showed the ammonia stress reduced galactosylation, sialylation, and fucosylation. Transcriptome analysis of the standard-aged cultures indicated the ammonia stress had a limited impact on the transcriptome, where few of the significant changes were directly related metabolite or glycosylation reactions. These results indicate that mechanisms used to alleviate ammonia stress are most likely controlled post-transcriptionally, and this is where future research should focus.

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance.

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A2A receptor (A2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A2AR C-terminus from 412 to 316 amino acids (A2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A2AR levels. As cAMP production is downstream of the first activation event-coupling of G protein to its receptor-investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gαs proteins, we characterize the association of A2AR and A2AΔ316R to Gαs with and without GDP or GTPγs using surface plasmon resonance (SPR). Gαs affinity for A2AR was greatest for apo-Gαs, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A2AR C-terminus (A2AΔ316R) decreased the affinity of the unliganded receptor for Gαs by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.

The Specificity of Downstream Signaling for A1 and A2AR Does Not Depend on the C-Terminus, Despite the Importance of This Domain in Downstream Signaling Strength.

Recent efforts to determine the high-resolution crystal structures for the adenosine receptors (A1R and A2AR) have utilized modifications to the native receptors in order to facilitate receptor crystallization and structure determination. One common modification is a truncation of the unstructured C-terminus, which has been utilized for all the adenosine receptor crystal structures obtained to date. Ligand binding for this truncated receptor has been shown to be similar to full-length receptor for A2AR. However, the C-terminus has been identified as a location for protein-protein interactions that may be critical for the physiological function of these important drug targets. We show that variants with A2AR C-terminal truncations lacked cAMP-linked signaling compared to the full-length receptor constructs transfected into mammalian cells (HEK-293). In addition, we show that in a humanized yeast system, the absence of the full-length C-terminus affected downstream signaling using a yeast MAPK response-based fluorescence assay, though full-length receptors showed native-like G-protein coupling. To further study the G protein coupling, we used this humanized yeast platform to explore coupling to human-yeast G-protein chimeras in a cellular context. Although the C-terminus was essential for Gα protein-associated signaling, chimeras of A1R with a C-terminus of A2AR coupled to the A1R-specific Gα (i.e., Gαi1 versus Gαs). This surprising result suggests that the C-terminus is important in the signaling strength, but not specificity, of the Gα protein interaction. This result has further implications in drug discovery, both in enabling the experimental use of chimeras for ligand design, and in the cautious interpretation of structure-based drug design using truncated receptors.

Membrane cholesterol depletion reduces downstream signaling activity of the adenosine A2A receptor.

Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, indirectly via modifications to the membrane, or via both mechanisms is not well understood. High-resolution crystal structures of GPCRs have identified bound cholesterols; based on a ß2-adrenergic receptor (ß2AR) structure bound to cholesterol and the presence of conserved amino acids in class A receptors, the cholesterol consensus motif (CCM) was identified. Here in mammalian cells expressing the adenosine A2A receptor (A2AR), ligand dependent production of cAMP is reduced following membrane cholesterol depletion with methyl-beta-cyclodextrin (MßCD), indicating that A2AR signaling is dependent on cholesterol. In contrast, ligand binding is not dependent on cholesterol depletion. All-atom molecular simulations suggest that cholesterol interacts specifically with the CCM when the receptor is in an active state, but not when in an inactive state. Taken together, the data support a model of receptor state-dependent binding between cholesterol and the CCM, which could facilitate both G-protein coupling and downstream signaling of A2AR.

CSF levels of oligomeric alpha-synuclein and beta-amyloid as biomarkers for neurodegenerative disease.

Protein misfolding and aggregation is a critically important feature in many devastating neurodegenerative diseases, therefore characterization of the CSF concentration profiles of selected key forms and morphologies of proteins involved in these diseases, including ß-amyloid (Aß) and α-synuclein (a-syn), can be an effective diagnostic assay for these diseases. CSF levels of tau and Aß have been shown to have great promise as biomarkers for Alzheimer's disease. However since the onset and progression of many neurodegenerative diseases have been strongly correlated with the presence of soluble oligomeric aggregates of proteins including various Aß and a-syn aggregate species, specific detection and quantification of levels of each of these different toxic protein species in CSF may provide a simple and accurate means to presymptomatically diagnose and distinguish between these diseases. Here we show that the presence of different protein morphologies in human CSF samples can be readily detected using highly selective morphology specific reagents in conjunction with a sensitive electronic biosensor. We further show that these morphology specific reagents can readily distinguish between post-mortem CSF samples from AD, PD and cognitively normal sources. These studies suggest that detection of specific oligomeric aggregate species holds great promise as sensitive biomarkers for neurodegenerative disease.