Synthesis of vitamin D in the skin in response to ultraviolet light is the main determinant of vitamin D status in man and it is therefore surprising that rickets and osteomalacia, clinical signs of vitamin D deficiency, remain common in tropical and subtropical countries. Skin pigmentation can reduce vitamin D formation but this is a negligible limitation in people exposed to abundant ultraviolet light. Earlier studies in animals and man suggested that another environmental factor, the low calcium/high cereal diet typical of susceptible populations, might affect the efficiency of vitamin D utilization. We show here in rats that the rate of inactivation of vitamin D in the liver is increased by calcium deprivation. The effect is mediated by 1,25-dihydroxyvitamin D, produced in response to secondary hyperparathyroidism, which promotes hepatic conversion of vitamin D to polar inactivation products that are excreted in bile. This finding has widespread implications both for understanding the pathogenesis of endemic rickets and in that it provides a unifying mechanism for the development of vitamin D deficiency in many clinical disorders.

G protein-coupled receptor kinases (GRKs) mediate desensitization of agonist-occupied G protein-coupled receptors (GPCRs). Here we report that GRK5 contains a DNA-binding nuclear localization sequence (NLS) and that its nuclear localization is regulated by GPCR activation, results that suggest potential nuclear functions for GRK5. As assessed by fluorescence confocal microscopy, transfected and endogenous GRK5 is present in the nuclei of HEp2 cells. Mutation of basic residues in the catalytic domain of GRK5 (between amino acids 388 and 395) results in the nuclear exclusion of the mutant enzyme (GRK5 ⌬NLS ), demonstrating that GRK5 contains a functional NLS. The nuclear localization of GRK5 is subject to dynamic regulation. Calcium ionophore treatment or activation of Gq-coupled muscarinic-M3 receptors promotes the nuclear export of the kinase in a Ca 2؉ /calmodulin (Ca 2؉ /CaM)-dependent fashion. Ca 2؉ /CaM binding to the N-terminal CaM binding site of GRK5 mediates this effect. Furthermore, GRK5, but not GRK5 ⌬NLS or GRK2, binds specifically and directly to DNA in vitro. Consistent with their presence in the nuclei of transfected cells, all the GRK4, but not GRK2, subfamily members contain putative NLSs. These results suggest that the GRK4 subfamily of GRKs may play a signaling role in the nucleus and that GRK4 and GRK2 subfamily members perform divergent cellular functions.G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine protein kinases that phosphorylate agonist-bound, activated, G protein-coupled receptors (GPCRs). GRK-mediated GPCR phosphorylation initiates ␤-arrestin binding, receptor uncoupling from the G protein, and targeting of the ␤-arrestin-receptor complex to a clathrin-coated pit for internalization. The receptor may then be degraded or returned to the cell surface for a further round of signaling (reviewed in reference 4).Seven mammalian GRKs have been identified, which are divided into three subfamilies on the basis of sequence homology and the regulatory mechanisms controlling their activity (reviewed in reference 24): the GRK1-like subfamily, GRK1 (or rhodopsin kinase) and GRK7; the GRK2-like subfamily, GRK2 (␤-adrenergic kinase) and GRK3 (␤-adrenergic kinase 2); and the GRK4-like subfamily, GRK4, GRK5, and GRK6. Four splice variants of GRK4 (␣, ␤, ␥, and ␦) and three splice variants of GRK6 (A, B, and C) have been identified (24).GRKs are regulated by several mechanisms, including modulation of their subcellular localization, kinase activity, and expression (reviewed in reference 21). In many instances, regulation of GRK activity is subfamily specific. PKC phosphorylation results in activation of GRK2 but inhibition of GRK5 activity (21). Additionally, the three GRK subfamilies display differential affinities for calcium binding proteins. GRK1 binds Ca 2ϩ /recoverin, and GRK4␣, GRK5, and GRK6A, -B and -C bind Ca 2ϩ /calmodulin (Ca 2ϩ /CaM) with high affinity (reviewed in reference 32). In contrast, GRK2 exhibits an approximately 40-fold lower affinity for C...

G protein-coupled receptor kinases (GRKs) act to desensitize G protein-coupled receptors (GPCRs). In addition to this role at the plasma membrane, a nuclear function for GRK5, a member of the GRK4 subfamily of GRKs, has been reported. GRK5 phosphorylates and promotes the nuclear export of the histone deacetylase, HDAC5. Here we demonstrate that the possession of a nuclear localization sequence (NLS) is a common feature of GRK4 subfamily members (GRKs 4, 5 and 6). However, the location of the NLS and the ability of these GRKs to bind DNA in vitro are different. The NLSs of GRK5 and 6 bind DNA in vitro, whilst the NLS of GRK4 does not. Using mutants of GRK5 we identify the regions of GRK5 required for DNA-binding in vitro and nuclear localization in cells. The DNA-binding ability of GRK5 requires both the NLS and an N-terminal calmodulin (CaM)-binding site. A functional nuclear export sequence (NES), required for CaM-dependent nuclear export of the kinase, is also identified. Based on our observations we propose a model to explain how nuclear localization of GRK5 may be regulated. Notably, the nuclear localization of GRK5 and 6 is differentially regulated. These results suggest subfamily specific nuclear functions for the GRK4 subfamily members. Identification of GRK specific small molecule inhibitors of nuclear localization and/or function for the GRK4 subfamily may thus be an achievable goal.