Limits...
Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors

View Article: PubMed Central - PubMed

ABSTRACT

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β2-adrenergic receptor (β2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the β2AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the β2AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.

No MeSH data available.


Related in: MedlinePlus

Homology-based representation of the positioning of the FlAsH tag in three class A G protein-coupled receptors. (A) Homology model of the hβ2AR (P07550-1) based on PDB identifier: 2rh1A with truncated C-tail. Positions highlighted in orange correspond to the first position, in blue the second position, and in green the third position within each respective loop structure. (B) Homology model of the human angiotensin II type 1 receptor (P30556-1) modeled upon the existing crystal structure with PBD accession 4yayA, the C-tail was then truncated (25). The ICL3 p3 biosensor is shown in green. (C) Homology model of the human FP (P43088-1) based on the PBD ID: 3emlA. Insertion of the TC tag in ICL3 position 4 is shown in red. (D) Superimposing the models of the hβ2AR, AT1R, and FP. Overlay of three receptors reveals the relative similarities in the transmembrane domains and differences in the cytoplasmic regions. Approximately 20 residues were removed from the N-terminus and the C-terminus was truncated to facilitate the visualization of the overall structure. Inset shows expanded versions of ICL2 (left) or ICL3 (right). The I-TASSER models (26, 27) were exported into PyMOL where the CCPGCC motifs were inserted at their respective positions and color coded to facilitate the visualization of the positioning of the FlAsH tags.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383666&req=5

Figure 7: Homology-based representation of the positioning of the FlAsH tag in three class A G protein-coupled receptors. (A) Homology model of the hβ2AR (P07550-1) based on PDB identifier: 2rh1A with truncated C-tail. Positions highlighted in orange correspond to the first position, in blue the second position, and in green the third position within each respective loop structure. (B) Homology model of the human angiotensin II type 1 receptor (P30556-1) modeled upon the existing crystal structure with PBD accession 4yayA, the C-tail was then truncated (25). The ICL3 p3 biosensor is shown in green. (C) Homology model of the human FP (P43088-1) based on the PBD ID: 3emlA. Insertion of the TC tag in ICL3 position 4 is shown in red. (D) Superimposing the models of the hβ2AR, AT1R, and FP. Overlay of three receptors reveals the relative similarities in the transmembrane domains and differences in the cytoplasmic regions. Approximately 20 residues were removed from the N-terminus and the C-terminus was truncated to facilitate the visualization of the overall structure. Inset shows expanded versions of ICL2 (left) or ICL3 (right). The I-TASSER models (26, 27) were exported into PyMOL where the CCPGCC motifs were inserted at their respective positions and color coded to facilitate the visualization of the positioning of the FlAsH tags.

Mentions: Crystal structures offer snapshot images of receptor structure that can be complemented using more dynamic measures such as RET approaches. Kobilka and coworkers reported that transmembrane domain VI experiences a 14 Å outward movement when comparing the inactive carazolol bound β2AR versus the active-Gαs bound crystal structure (24). We show here that three different GPCRs show distinct patterns of BRET in response to ligand even when biosensors are placed in similar positions (Figure 7).


Distinct Conformational Dynamics of Three G Protein-Coupled Receptors Measured Using FlAsH-BRET Biosensors
Homology-based representation of the positioning of the FlAsH tag in three class A G protein-coupled receptors. (A) Homology model of the hβ2AR (P07550-1) based on PDB identifier: 2rh1A with truncated C-tail. Positions highlighted in orange correspond to the first position, in blue the second position, and in green the third position within each respective loop structure. (B) Homology model of the human angiotensin II type 1 receptor (P30556-1) modeled upon the existing crystal structure with PBD accession 4yayA, the C-tail was then truncated (25). The ICL3 p3 biosensor is shown in green. (C) Homology model of the human FP (P43088-1) based on the PBD ID: 3emlA. Insertion of the TC tag in ICL3 position 4 is shown in red. (D) Superimposing the models of the hβ2AR, AT1R, and FP. Overlay of three receptors reveals the relative similarities in the transmembrane domains and differences in the cytoplasmic regions. Approximately 20 residues were removed from the N-terminus and the C-terminus was truncated to facilitate the visualization of the overall structure. Inset shows expanded versions of ICL2 (left) or ICL3 (right). The I-TASSER models (26, 27) were exported into PyMOL where the CCPGCC motifs were inserted at their respective positions and color coded to facilitate the visualization of the positioning of the FlAsH tags.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383666&req=5

Figure 7: Homology-based representation of the positioning of the FlAsH tag in three class A G protein-coupled receptors. (A) Homology model of the hβ2AR (P07550-1) based on PDB identifier: 2rh1A with truncated C-tail. Positions highlighted in orange correspond to the first position, in blue the second position, and in green the third position within each respective loop structure. (B) Homology model of the human angiotensin II type 1 receptor (P30556-1) modeled upon the existing crystal structure with PBD accession 4yayA, the C-tail was then truncated (25). The ICL3 p3 biosensor is shown in green. (C) Homology model of the human FP (P43088-1) based on the PBD ID: 3emlA. Insertion of the TC tag in ICL3 position 4 is shown in red. (D) Superimposing the models of the hβ2AR, AT1R, and FP. Overlay of three receptors reveals the relative similarities in the transmembrane domains and differences in the cytoplasmic regions. Approximately 20 residues were removed from the N-terminus and the C-terminus was truncated to facilitate the visualization of the overall structure. Inset shows expanded versions of ICL2 (left) or ICL3 (right). The I-TASSER models (26, 27) were exported into PyMOL where the CCPGCC motifs were inserted at their respective positions and color coded to facilitate the visualization of the positioning of the FlAsH tags.
Mentions: Crystal structures offer snapshot images of receptor structure that can be complemented using more dynamic measures such as RET approaches. Kobilka and coworkers reported that transmembrane domain VI experiences a 14 Å outward movement when comparing the inactive carazolol bound β2AR versus the active-Gαs bound crystal structure (24). We show here that three different GPCRs show distinct patterns of BRET in response to ligand even when biosensors are placed in similar positions (Figure 7).

View Article: PubMed Central - PubMed

ABSTRACT

A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the β2-adrenergic receptor (β2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the β2AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the β2AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.

No MeSH data available.


Related in: MedlinePlus