Historical Perspective of the G Protein-Coupled Receptor Kinase Family

Abstract

Agonist activation of G protein-coupled receptors promotes sequential interaction of the receptor with heterotrimeric G proteins, G protein-coupled receptor kinases (GRKs), and arrestins. GRKs play a central role in mediating the switch from G protein to arrestin interaction and thereby control processes such as receptor desensitization and trafficking and arrestin-mediated signaling. In this review, I provide a historical perspective on some of the early studies that identified the family of GRKs with a primary focus on the non-visual GRKs. These studies included identification, purification, and cloning of the β-adrenergic receptor kinase in the mid- to late-1980s and subsequent cloning and characterization of additional members of the GRK family. This helped to lay the groundwork for ensuing work focused on understanding the structure and function of these important enzymes.

Keywords: GPCR; GRK; arrestins; phosphorylation; signaling.

Figure 1

S49 kin⁻ cell extract phosphorylation of purified β₂AR. S49 lymphoma kin⁻ cells were lysed, centrifuged, and the supernatant was incubated with MgCl₂, radiolabeled ATP, purified hamster lung β₂AR reconstituted in phospholipid vesicles, and either no ligand (lane 1), 10 μM isoproterenol (ISO) (lane 2), or 10 μM ISO plus 20 μM alprenolol (ALP) (lane 3) for 30 min at 30 °C. The samples were centrifuged, and the pellets were washed, solubilized, and the β₂AR was purified on a small alprenolol affinity column and then run on SDS-PAGE and exposed to film.

Figure 2

Cloning of a β-adrenergic receptor kinase (βARK) cDNA from a bovine brain cDNA library. One nmol of purified βARK was treated with CNBr, and the resulting peptides were separated and sequenced. A total of eight distinct peptide sequences were identified, with two being used in the design of oligonucleotide probes for cloning. (A) Amino acid sequence of two peptides that were consistently observed in multiple βARK preparations and the sequences of the oligonucleotide probes that were used to hybridize with cDNA libraries. The underlined residues in peptide #2 were found to be incorrect. (B) A Southern blot of several clones (3A, 3B, 4, and 13) isolated from the bovine brain cDNA library using radiolabeled oligo 2526 derived from peptide #1. Two colonies from each clone were digested with Eco RI, transferred to nitrocellulose, and probed with radiolabeled oligos derived from peptide #1 (2526) or peptide #2 (2288). The clones were sequenced, and clones 3A and 4 were found to contain a complete open reading frame. I—inosine.