G Protein-coupled Receptor Kinase 3 (GRK3) Gene Disruption Leads to Loss of Odorant Receptor Desensitization

Abstract

G protein-coupled receptor kinases (GRKs) 2 and 3 (␤-adrenergic receptor kinases 1 and 2 (␤ARK1 and -2)) mediate the agonist-dependent phosphorylation and uncoupling of many G protein-coupled receptors. These two members of the GRK family share a high degree of sequence homology and show overlapping patterns of substrate specificity in vitro. To define their physiological roles in vivo we have generated mice that carry targeted disruption of these genes. In contrast to GRK2-deficient mice, which die in utero (Jaber, M., Koch, W. J., Rockman, H., Smith, B., Bond, R. A., Sulik, K. K., Ross, J. JR., Lefkowitz, R. J. Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974 -12979), GRK3 deletion allows for normal embryonic and postnatal development. GRK3 is expressed to a high degree in the olfactory epithelium, where GRK2 is absent. Here we report that cilia preparations derived from GRK3-deficient mice lack the fast agonist-induced desensitization normally seen after odorant stimulation. Moreover, total second messenger (cAMP) generation in these cilia preparations following odorant stimulation is markedly reduced when compared with preparations from wildtype littermates. This reduction in the ability to generate cAMP is evident even in the presence of nonodorant receptor stimuli (GTP␥S and forskolin), suggesting a compensatory dampening of the G protein-adenylyl cyclase system in the GRK3 (؊/؊) mice in the olfactory epithelium. These findings demonstrate the requirement of GRK3 for odorant-induced desensitization of cAMP responses.Many G protein-coupled receptors (GPCRs) 1 show diminished ability to signal and couple to G proteins after prolonged or repeated agonist stimulation. This phenomenon, referred to as agonist-mediated desensitization, occurs very rapidly and is initiated via receptor phosphorylation by G protein-coupled receptor kinases (GRKs) that serve to uncouple the receptor from its G protein (1).Whereas the function of these proteins has been mostly studied in vitro and in tissue culture the physiological relevance of the mechanisms initiated by them have just begun to be explored. While there is some evidence of substrate specificity among the different members of the GRK family, most show activity toward a wide variety of agonist-occupied receptors in vitro. This, in addition to their ubiquitous tissue expression, has made it difficult to precisely determine the role of the GRKs in vivo. To clarify the physiological role of the individual members of this family, we have generated mice that carry targeted disruptions of the GRK2 or GRK3 (␤ARK2) genes. GRK2 deletion is embryonically lethal as homozygous mice die in utero before gestational day 15.5 of severe cardiac malformations (2). Whereas GRKs 2 and 3 show 81% amino acid identity (3) and an overlapping pattern of tissue expression (4), GRK3 apparently is not able to compensate for the loss of GRK2 in embryogenesis. In most tissues examined GRK2 is the predominant form. However, in the olfactory epithelium GRK2 is virtually a...

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