Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs.     

Rate of thymidine incorporation and incidence of parenchymal cell division in adult rat hypophyseal cell cultures. Effect of thyroliberin and somatostatin

Cell suspensions derived from adult rat anterior pituitary glands were cultured for up to eight days. Prolactin immunoreactivity and/or tritiated thymidine incorporation into DNA of cell nuclei were demonstrated in cells with and without thyroliberin (TRH) and somatostatin (SRIF) treatment. It has been established that (a) TRH, which is effective in releasing both thyrotropin and prolactin, may stimulate cell proliferation in other than its target cells; that (b) SRIF has no effect on lactotropic cell proliferation and augments thymidine incorporation into DNA of unidentified cells; that (c) immunoreactive lactotropic cells with tritium-labelled nuclei are present in each culture, independent of hypothalamic hormone treatments.     

Stimulation by insulin of DNA synthesis in cultured mouse fibroblasts]

With the aid of autoradiography, the effect of insulin on entering S- from G1-period of the mitotic cycle and on the rate of DNA synthesis of the mouse fibroblasts (L), was studied,--in the cells incubated for 24 hr in serum-free medium. In these conditions the cells were temporarily blocked in G1-period. Insulin (100 mcU/ml) increased by 1.5-fold the amount of cells in S-period as well as caused a marked stimulation of DNA synthesis.     

Obtaining and morphological characteristics of primary monolayer cell cultures of human somatotropinomas]

This paper describes the development of method of preparing cell suspension obtained from surgical material of patients with pituitary adenomas and acromegaly as well as the procedure of subsequent long-term cultivation in monolayers of the cells isolated. As judged by visual inspection and measurement of growth hormone and prolactin secretion, tumor pituitary cells kept viability and functional activity for at least 6 days of growing in vitro. Immunocytochemical visualization of somato- and lactotrophs of the same histological preparations permitted us to show that vast majority of cultured cells is represented by somatotrophs; however, a small portion of cell population is represented by lactotrophs and lactosomatotrophs. The peculiarities of cytoarchitectonics in two types of cell cultures of human somatotropinomas were studied.     

Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647

Recoverin belongs to the superfamily of EF-hand Ca2+-binding proteins and operates as a Ca2+-sensor in vertebrate photoreceptor cells, where it regulates the activity of rhodopsin kinase GRK1 in a Ca2+-dependent manner. Ca2+-dependent conformational changes in recoverin are allosterically controlled by the covalently attached myristoyl group. The amino acid sequence of recoverin harbors a unique cysteine at position 38. The cysteine can be modified by the fluorescent dye Alexa647 using a maleimide-thiol coupling step. Introduction of Alexa647 into recoverin did not disturb the biological function of recoverin, as it can regulate rhodopsin kinase activity like unlabeled recoverin. Performance of the Ca2+-myristoyl switch of labeled recoverin was monitored by Ca2+-dependent association with immobilized lipids using surface plasmon resonance spectroscopy. When the Ca2+-concentration was varied, labeled myristoylated recoverin showed a 37%-change in fluorescence emission and a 34%-change in excitation intensity, emission and excitation maxima shifted by 6 and 18 nm, respectively. In contrast, labeled nonmyristoylated recoverin exhibited only minimal changes. Time-resolved fluorescence measurements showed biexponentiell fluorescence decay, in which the slower time constant of 2 ns was specifically influenced by Ca2+-induced conformational changes. A similar influence on the slower time constant was observed with the recoverin mutant RecE85Q that has a disabled EF-hand 2, but no such influence was detected with the mutant RecE121Q (EF-hand 3 is nonfunctional) that contains the myristoyl group in a clamped position. We conclude from our results that Alexa647 bound to cysteine 38 can monitor the conformational transition in recoverin that is under control of the myristoyl group.     

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.     

Visual transduction and calcium ions

A general scheme of visual induction and its regulation in mammalian retinal rods and the main signal proteins involved in the functioning of the visual signaling apparatus were considered. The role of calcium ions and calcium-binding proteins in the switching off of the visual signal and the transition of the photoreceptor cell from a photoexcited to a "dark" state was discussed in detail.     

Calcium modifies the effects of thyroliberin and somatostatin on hormone release from cell cultures of the rat anterior pituitary

Role of calcium (Ca2+) in the effects of thyroliberin (TRH) and somatostatin (SRIF) on the release of growth hormone (GH), prolactin (PRL) and thyroid stimulating hormone (TSH) from the rat adenohypophyseal cells in primary monolayer cultures has been studied. Decrease of extracellular Ca2+ diminished the stimulatory effects of TRH on TSH and PRL release. Ca2+ is also an important factor in the mechanism of SRIF action. Data obtained in the experiments with high Ca2+ levels in the medium indicate that some antagonistic interrelationship exists between Ca2+ and SRIF. These results suggest that the participation of cAMP alone is not sufficient for stimulus-secretion coupling. Another messenger, namely Ca2+, is necessary for the effects of hypothalamic hormones. On the other hand, the contribution of Ca2+ to the secretory process in mammotrophs, somatotrophs and thyrotrophs is not equal. PRL and TSH secretion is more dependent on the presence of extracellular Ca2+ than the release of GH.     

DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures. Effects of thyroliberin, bromocriptine and somatostatin

Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154). The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells. TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs. TRH only weakly counteracted the noticeable inhibitory effect of CB 154 (0.75 microM/l) on thymidine incorporation into lactotrophs. SRIF (20 ng/ml) inhibited DNA synthesis in lactotrophs to a lesser extent than CB 154. The combination of methods used in this paper may be useful for studying the selective effects of regulators on the proliferative activity of various pituitary cell types in vivo and in culture.