Probing the constitutive activity among dopamine D1 and D5 receptors and their mutants.

Dopamine D1 and D5 receptors are prototypical cell-surface seven-transmembrane (TM) G protein-coupled receptors (GPCRs) mediating elevation of intracellular cAMP levels. The high level of constitutive activity of D5 receptor mediating intracellular cAMP production is one of the functional hallmarks distinguishing the closely related D1-like dopaminergic subtypes (D1 and D5). D1-like subtypes share over 80% identity within their TM regions. Thus, D1 and D5 receptors can serve as unparalleled and useful molecular tools to gain structural and mechanistic insights into subtype-specific determinants regulating GPCR constitutive activation and inverse agonism. A method has been developed that relies on the use of transfected human embryonic kidney 293 cells with wild-type (WT), epitope-tagged, chimeric, truncated, and mutant forms of mammalian D1 and D5 receptors using a modified DNA and calcium phosphate precipitation procedure. Receptor expression levels are quantified by a radioligand binding using [(3)H]-SCH23390, a D1-like selective drug. Regulation of ligand-independent and dependent activity of WT and mutated D1 and D5 receptors is determined by whole cell cAMP assays using metabolic [(3)H]-adenine labeling and sequential purification radiolabeled nucleotides over Dowex and alumina resin columns. Results on the regulation of D1 and D5 constitutive activity are presented here. Our studies indicate that dopamine-mediated D5 receptor stimulation in a dose-dependent manner is not always detectable, suggesting that D5 receptors can exist in a "locked" constitutively activated state. This "locked" constitutively active state of D5 receptor is not linked to aberrant high receptor expression levels or cell behavior, as D1 receptor function remains essentially unchanged in these cells. In fact, we show that phorbol ester treatment of cells harboring "locked" constitutively active D5 receptors abrogates constitutive activation of D5R to allow its stimulation by dopamine in a dose-dependent manner.

B1 integrin/Fak/Src signaling in intestinal epithelial crypt cell survival: integration of complex regulatory mechanisms.

The molecular determinants which dictate survival and apoptosis/anoikis in human intestinal crypt cells remain to be fully understood. To this effect, the roles of beta1 integrin/Fak/Src signaling to the PI3-K/Akt-1, MEK/Erk, and p38 pathways, were investigated. The regulation of six Bcl-2 homologs (Bcl-2, Mcl-1, Bcl-X(L), Bax, Bak, Bad) was likewise analyzed. We report that: (1) Anoikis causes a down-activation of Fak, Src, Akt-1 and Erk1/2, a loss of Fak-Src association, and a sustained/enhanced activation of p38beta, which is required as apoptosis/anoikis driver; (2) PI3-K/Akt-1 up-regulates the expression of Bcl-X(L) and Mcl-1, down-regulates Bax and Bak, drives Bad phosphorylation (both serine112/136 residues) and antagonizes p38beta activation; (3) MEK/Erk up-regulates Bcl-2, drives Bad phosphorylation (serine112 residue), but does not antagonize p38bactivation; (4) PI3-K/Akt-1 is required for survival, whereas MEK/Erk is not; (5) Src acts as a cornerstone in the engagement of both pathways by beta1 integrins/Fak, and is crucial for survival; and (6) beta1 integrins/Fak and/or Src regulate Bcl-2 homologs as both PI3-K/Atk-1 and MEK/Erk combined. Hence, beta1 integrin/Fak/Src signaling translates into integrated mediating functions of p38beta activation and regulation of Bcl-2 homologs by PI3-K/Akt-1 and MEK/Erk, consequently determining their requirement (or not) for survival.

Fak/Src signaling in human intestinal epithelial cell survival and anoikis: differentiation state-specific uncoupling with the PI3-K/Akt-1 and MEK/Erk pathways.

Human intestinal epithelial cell survival and anoikis are distinctively regulated according to the state of differentiation. In the present study, we analyzed the roles of focal adhesion kinase (Fak)/Src signaling to the PI3-K/Akt-1 and mitogen-activated protein kinase (MEK)/extracellular regulated kinases (Erk) pathways, within the context of such differentiation-state distinctions. Anoikis was induced by inhibition of beta1 integrins (antibody blocking), inhibition of Fak (pharmacologic inhibition or overexpression of dominant negative mutants), or by maintaining cells in suspension. Activation parameters of Fak, Src, Akt-1, and Erk1/2 were analyzed. Activities of Src, Akt-1, or Erk1/2 were also blocked by pharmacological inhibition or by overexpression of dominant-negative mutants. We report that: (1) the loss or inhibition of beta1 integrin binding activity causes anoikis and results in a down-activation of Fak, Src, Akt-1, and Erk1/2 in both undifferentiated, and differentiated cells; (2) the inhibition of Fak likewise causes anoikis and a down-activation of Src, Akt-1, and Erk1/2, regardless of the differentiation state; (3) Src, PI3-K/Akt-1, and MEK/Erk contribute to the survival of differentiated cells, whereas MEK/Erk does not play a role in the survival of undifferentiated ones; (4) the inhibition/loss of beta1 integrin binding and/or Fak activity results in a loss of Src engagement with Fak, regardless of the state of differentiation; and (5) Src contributes to the activation of both the PI3-K/Akt-1 and MEK/Erk pathways in undifferentiated cells, but does not influence PI3-K/Akt-1 in differentiated ones. Hence, Fak/Src signaling to the PI3-K/Akt-1 and MEK/Erk pathways undergoes a differentiation state-specific uncoupling which ultimately reflects upon the selective engagement of these same pathways in the mediation of intestinal epithelial cell survival.