In the present study, we identified β2AR-interacting proteins in rat heart cytosol by using the full-length β2ARs reconstituted in the plasma membrane-mimicking HDL particles. To our knowledge, this is the first comprehensive study to investigate GPCR-interacting proteins in the heart or any other primary tissues other than brain. The advantage of reconstituting GPCRs in the HDL particles is that GPCRs are more stable and in a more physiological conformation than detergent-solubilized GPCRs. Furthermore, this approach overcomes the low endogenous expression of the receptor in the heart by using large amount of β2AR•rHDL as bait.
Various methods have been used to screen for direct and indirect binding partners of GPCRs. Among those, the affinity isolation/mass spectrometry-based proteomic approach allows the capture and analysis of larger proteome units of protein complexes and can be used for isolating and purifying complexes from cellular and tissue preparations 
. However, the proteomic analysis of GPCRs has been challenging due to low endogenous expression levels and hydrophobicity of GPCRs. To date, identification of interacting proteins in native tissue has been successful for few GPCRs; including, mGluR5, 5-HT receptors (5-HT-2a, 5HT-2c, and 5-HT4a), and α2
. All of these GPCRs were studied in brain tissue where GPCRs and their binding partners are highly expressed, enabling isolation of sufficient quantities of the receptor and associated proteins. Furthermore, studies with 5-HT receptors and α2
B-AR used c-terminal peptides of the receptors (not the full-length GPCR) as baits 
. Therefore, those studies cannot identify binding partners that interact with GPCR domains outside of the c-terminus. The present study successfully used full-length β2
AR as bait and identified binding partners from heart tissue, where the expression level of endogenous β2
AR is very low.
Although the present study identified β2
AR-interacting proteins from heart cytosol, there are limitations. First, β2
AR-interacting membrane proteins cannot be purified because β2
AR is trapped in the rHDL and detergents cannot be used to solubilize the membrane proteins. GPCRs interact with membrane proteins as well as cytosolic proteins. β2
AR is also known to interact various membrane proteins (Table S3
), but we could not purify these proteins due to the limitations of the system. Second, the results of the present study do not represent proteins that bind to β2
AR with post-translational modifications (PTMs). GPCRs are known to undergo various PTMs including phosphorylation, ubiquitination, glycosylation and nitrosylation 
. However, β2
AR purified from insect cells does not contain the same PTMs vs. β2
AR from mammalian cells. Therefore, proteins that are known to interact with phosphorylated β2
AR (e.g. β-arrestins) 
were not identified in this study. Lastly, as expected, not all previously known β2
AR interacting proteins were identified in our search (Table S3
). This may be due to the intrinsic limitation of mass spectrometry-based protein identification (false-negative detection), low binding affinity of those proteins to β2
AR, or the artificial environment of β2
AR•rHDL. Additional studies are required to overcome these limitations; however, we believe that the described method represents an improvement on previously described methods for identifying GPCR-interacting proteins.
Bioinformatic analyses of β2AR•rHDL pull-downs showed distinct protein profiles compared to control pull-downs (). Functional analysis indicated that a higher percentage of proteins from β2AR•rHDL pull-downs are involved in cell signaling and protein trafficking when compared with controls (), suggesting that the identified proteins are not the result of non-specific binding. Canonical pathway analysis used the list of identified proteins to predict relevant signaling pathways and confirmed the difference between β2AR•rHDL pull-downs and control pull-down. The majority of pathways from β2AR•rHDL pull-downs are signal-transduction related pathways; whereas, most of the top 15 pathways from control pull-downs are related to metabolic proteins that are enriched in the heart (). In addition to the known β2AR signaling pathways in the heart (eg. cardiac β-adrenergic signaling, protein ubiquitination, clathrin-mediated endocytosis and G beta gamma signaling), the present study suggests the involvement of the β2AR in novel signaling pathways; such as AMPK signaling, PI3K/AKT signaling and integrin signaling pathways (). β2AR interaction with selected proteins identified in the β2AR•rHDL pull-downs were confirmed by co-immunoprecipitation and Western Blot analysis () indicating that the identified proteins are not false-positives. The role of these novel signaling pathways in β2AR function in heart physiology and pathology warrants further investigation. Taken together, these bioinformatic analyses confirm the utility of using of GPCR•rHDL as an experimental system to identify GPCR-interacting proteins.