Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases.

R.T Premont,W.J Koch,J Inglese,R.J Lefkowitz

doi:10.1016/s0021-9258(17)37451-3

R.T Premont, W.J Koch + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)37451-3

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Abstract

A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner and phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function.

Highlights

A novel member of the family of G protein-coupled with an arrestin proteiwn,hich prevents the furthceorupling of receptor kinases(GRKS),named GRKS,has been cloned the receptor to G proteins, and results ina desensitized from bovine taste epithelium
GRKS mRNA is foundmost diate tEs activation-dependent phosphorylation of receptors abundantly in lung, heart, retina, and lingual epithehave only recently been characterized at the molecular level lium, butis expressed very little in brain, liver, kidne(y6,).Two widely distributed "0-adrenergic receptor kinase" isoor testis
The GRK5 and GRK6 kinergic receptor kinase and rhodopsin kinase, but rantahsercDNAs were recently cloned from human heart, and the appearstoassociatewithmembranesconstitutively

Summary

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Howard Hughes MedicalInstitute, Depts. of Medicine and Biochemis- Technologies,Inc.) [20].cDNA from 10 ng of bovine circumvallate patry, Box 3821,Duke University Medical Center, Durham, NC 27710. Purified enzymewas incubated with 100 p~ ATP (10 dpdfmol) as Individual 5' and 3' end cassettes were amplified with TaqDNApo- described, but the reaction was terminated by passage through a Biolymerase [20]using the full-length cDNA as template. The supernatant fewest protein contaminants were pooled (20 ml), diluted with buffer A fractions were removed and supplemented with 25 pmol of rhodopsin, to below 100 m~ NaC1, and applied to a 10-mlcolumn of heparin- while pelleted membranes were resuspended in the original volume of Sepharose (Pharmacia)at 1ml/min. Proximately800 m~ NaCl werepooled (20 ml), diluted 4-foldwith buffer Peptide Phosphorylation Assay-The PARK and rhodopsin kinase. Recombinant bovine PARK1 volume with autophosphorylation assay buffer, containing 500 pg/ml and bovine rhodopsinkinase were purified from lysates of appropriately bovine serum albumin, for 15 min at 30 "C.Assays weretermi-nated by infected Sf9 cells byessentially similar procedures [13]. Curve-fitting was performed using the SigmaPlot program using single experiments, and parameters from replicate experiments were averaged

RESULTS

A Coornassie

DISCUSSION