Distribution analysis of nonsynonymous polymorphisms within the G-protein-coupled receptor gene family

The G-protein-coupled receptor (GPCR) superfamily is one of the largest classes of proteins in mammalian genomes. GPCRs mediate diverse physiological functions and are the targets of >50% of all clinical drugs. The sequencing of the human genome and large-scale polymorphism discovery efforts have established an abundant source of single nucleotide polymorphisms (SNPs), particularly those that result in a change in the encoded amino acids (cSNPs), many are of which in GPCRs. Although the majority of these cSNPs are assumed not to be disease-causing (nDCs), experimental data on their functional impact are lacking. Here, we have computationally analyzed the distribution of 454 cSNPs within the GPCR gene family and have found that disease-causing cSNPs (DCs) are overrepresented, whereas nDCs are underrepresented or neutral in transmembrane and extracellular loop domains, respectively. This finding reflects the relative importance of these domains to GPCR function and implies different biological characteristics for the two sets of human polymorphisms.     

The necessity for T cell help for human tonsil B cell responses to pokeweed mitogens: induction of DNA synthesis, immunoglobulin, and specific antibody production with a T cell helper factor produced with pokeweed mitogen.

Human B lymphocytes obtained from tonsils do not proliferate when stimulated with pokeweed mitogen. A soluble factor produced from T cells cultured with pokeweed mitogen stimulates B cells to synthesize DNA and differentiate into immunoglobulin producing cells. This PWM produced supernatant induced a PFC response to SRBC. The T cell supernatant activity is produced within 12 hr of stimulation in the presence of serum and without a requirement for T cell division. Optimal stimulation of B cells occurred at 7 to 9 days of culture. This helper factor activity eluted postalbumin from a column of Sephadex G-200. Insolubilized pokeweed mitogen was not mitogenic for B cells. The continuous presence of the lectin in culture was not required for B cell proliferation or for immunoglobulin synthesis.     

beta-adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells

-Adrenergic receptor (AR) activationand/or increases in cAMP regulate growth and proliferation of a varietyof cells and, in some cells, promote cell death. In the current studieswe addressed the mechanism of this growth reduction by examiningAR-mediated effects in the murine T-lymphoma cell line S49.Wild-type S49 cells, derived from immature thymocytes(CD4⁺/CD8⁺) undergo growth arrest andsubsequent death when treated with agents that increase cAMP levels(e.g., AR agonists, 8-bromo-cAMP, cholera toxin, forskolin).Morphological and biochemical criteria indicate that this cell death isa result of apoptosis. In cyc and kin S49cells, which lack G_s and functional protein kinase A(PKA), respectively, AR activation of G_s and cAMPaction via PKA are critical steps in this apoptotic pathway. S49 cellsthat overexpress Bcl-2 are resistant to cAMP-induced apoptosis. Weconclude that AR activation induces apoptosis in immature Tlymphocytes via G_s and PKA, while overexpression ofBcl-2 prevents cell death. AR/cAMP/PKA-mediated apoptosis mayprovide a means to control proliferation of immature T cells in vivo.

     

Retrovirally mediated transfer of a G protein-coupled receptor kinase (GRK) dominant-negative mutant enhances endogenous calcitonin receptor signaling in Chinese hamster ovary cells. GRK inhibition enhances expression of receptors and receptor mRNA

G protein-coupled receptor kinases (GRKs) initiate pathways leading to agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. However, the role of GRKs in modulation of signaling properties of native receptors has not been clearly defined. Here we addressed this question by generating Chinese hamster ovary (CHO) cells stably expressing a dominant-negative mutant of GRK2 (DN-GRK2), K220R, using retrovirally mediated gene transfer, and we assessed function of the endogenously expressed calcitonin (CT) receptors. We found that CT-mediated responses were prominently enhanced in CHO cells expressing DN-GRK2 compared with mock-infected control CHO cells with approximately 3-fold increases in CT-promoted cAMP production in whole cells and adenylyl cyclase activity in membrane fractions. CT-promoted phosphoinositide hydrolysis was also enhanced in DN-GRK2 cells. The number of CT receptors was increased approximately 3-fold in DN-GRK2 cells, as assessed by (125)I-salmon CT-specific binding, and this was associated with increased CT receptor mRNA levels. These results indicate that DN-GRK2 has multiple consequences for CT receptor signaling, but a primary effect is an increase in CT receptor mRNA and receptor number and, in turn, enhanced CT receptor signaling. As such, our findings provide a mechanistic basis for previous observations regarding agonist-promoted down-regulation of CT receptors and for resistance and escape from response to CT in vitro and in vivo. Moreover, the data suggest that blunting of receptor desensitization by DN-GRK2 blocks a GRK-mediated tonic inhibition of CT receptor expression and response. We speculate that GRKs play a similar role for other G protein-coupled receptors as well.     

Protein kinase C isozymes and the regulation of diverse cell responses

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.     

Cellular mechanisms of social attachment

Pharmacological studies in prairie voles have suggested that the neuropeptides oxytocin and vasopressin play important roles in behaviors associated with monogamy, including affiliation, paternal care, and pair bonding. Our laboratory has investigated the cellular and neuroendocrine mechanisms by which these peptides influence affiliative behavior and social attachment in prairie voles. Monogamous prairie voles have a higher density of oxytocin receptors in the nucleus accumbens than do nonmonogamous vole species; blockade of these receptors by site-specific injection of antagonist in the female prairie vole prevents partner preference formation. Prairie voles also have a higher density of vasopressin receptors in the ventral pallidal area, which is the major output of the nucleus accumbens, than montane voles. Both the nucleus accumbens and ventral pallidum are key relay nuclei in the brain circuits implicated in reward, such as the mesolimbic dopamine and opioid systems. Therefore, we hypothesize that oxytocin and vasopressin may be facilitating affiliation and social attachment in monogamous species by modulating these reward pathways.     

Subunit dissociation is the mechanism for hormonal activation of the Gs protein in native membranes

We have recently reported (Ransn?s, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.     

Regulation of phosphodiesterase and ornithine decarboxylase by cAMP is cell cycle independent.

Cyclic AMP (cAMP) causes growth arrest in G1 and induction of cAMP phosphodiesterase and decrease of ornithine decarboxylase in S49 mouse lymphoma cells. Dibutyryl cAMP treatment of partially synchronized cells causes similar changes in activities of both enzymes, regardless of position in the cell cycle. This suggests that cAMP regulation of these enzymes is not mediated by growth perturbation.