Antibodies are characterized by versatility in form and in function: they are great communicators in the immune system, they can be used to visualize obscure targets, and can stabilize otherwise transient molecular states. In 2011, Brian K. Kolbilka and Robert J. Lefkowitz utilized camelid antibodies to determine the structure of a previously evasive but ubiquitous receptor-- an experiment that helped them earn a Nobel Prize.
GPCR Pathway

Discovery of G Proteins

Lefkowitz’s journey began with the qualification of the adrenergic receptor as separate from its ligand: epinephrine. He accomplished this by attaching radioactive iodine to adrenocorticotropic hormone and examining its binding activity. Once the presence of an independent receptor was verified, it had to be isolated, purified, and studied. Despite the receptor’s low concentration in tissue, Lefkowitz was able to solubilize it effectively and purify the receptor using affinity chromatography with columns of receptor-specific ligand.

Once the first epinephrine receptor, βAR, was isolated and purified, Lefkowitz and his team set out to clone and sequence it, which resulted in the identification of GPCRs as a family of closely related proteins.

Robert J. Lefkowtiz and Brian K. Kolbilka then launched their investigation into the extensive family of G-protein coupled receptors (GPCR): a structurally related group of receptors that often serve as points of regulation for physiological activity due to their intrinsic activation of powerful signaling cascades. Though there are multiple types of GPCRs, they all share a few distinguishing characteristics:  

·  Cross the cellular membrane 7 times

 

·  Heterotrimeric (composed of three subunits)

·  Trigger signal transduction pathways

 

·  Upon activation, undergo a conformational change

Enter: Single Domain Antibodies
Nature GPCR single domain antibody crystallography

Lefkowitz and Kolbilka began with the human β2 adrenergic receptor, attempting to crystalize the protein in its inactivated state. In their pursuit, the researchers encountered a major obstacle: the receptor’s significant mobility. Kolbilka, Stevens, and Schertler developed and used single domain antibodies to stabilize the Gβ-subunit in a well-defined conformation; covalent agonist coupling. This allowed for the characterization and definition of the inactivated G protein coupled receptor, one of many accomplishments that earned Lefkowitz and Kolbilka their Nobel Prize.

Antibodies can help solve a variety of problems that stand between you and your research. Let us help you leverage them effectively. For single domain antibody development and consulting, contact ProSci – your antibody experts.

 

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