Taroh Iiri

Similar Structures and Shared Switch Mechanisms of the β2-Adrenoceptor and the Parathyroid Hormone Receptor Zn(II) BRIDGES BETWEEN HELICES III AND VI BLOCK ACTIVATION

The seven transmembrane helices of serpentine receptors comprise a conserved switch that relays signals from extracellular stimuli to heterotrimeric G proteins on the cytoplasmic face of the membrane. By substituting histidines for residues at the cytoplasmic ends of helices III and VI in retinal rhodopsin, we engineered a metal-binding site whose occupancy by Zn(II) prevented the receptor from activating a retinal G protein, Gt (Sheikh, S. P., Zvyaga, T. A., Lichtarge, O., Sakmar, T. P., and Bourne, H. R. (1996)Nature 383, 347–350). Now we report engineering of metal-binding sites bridging the cytoplasmic ends of these two helices in two other serpentine receptors, the β2-adrenoreceptor and the parathyroid hormone receptor; occupancy of the metal-binding site by Zn(II) markedly impairs the ability of each receptor to mediate ligand-dependent activation of Gs, the stimulatory regulator of adenylyl cyclase. We infer that these two receptors share with rhodopsin a common three-dimensional architecture and an activation switch that requires movement, relative to one another, of helices III and VI; these inferences are surprising in the case of the parathyroid hormone receptor, a receptor that contains seven stretches of hydrophobic sequence but whose amino acid sequence otherwise shows no apparent similarity to those of receptors in the rhodopsin family. These findings highlight the evolutionary conservation of the switch mechanism of serpentine receptors and help to constrain models of how the switch works.

Gαi Is Not Required for Chemotaxis Mediated by Gi-coupled Receptors

Pertussis toxin inhibits chemotaxis of neutrophils by preventing chemoattractant receptors from activating trimeric G proteins in the Gi subfamily. In HEK293 cells expressing recombinant receptors, directional migration toward appropriate agonist ligands requires release of free G protein βγ subunits and can be triggered by agonists for receptors coupled to Gi but not by agonists for receptors coupled to two other G proteins, Gs and Gq. Because activation of any G protein presumably releases free Gβγ, we tested the hypothesis that chemotaxis also requires activated α subunits (Gαi) of Gi proteins. HEK293 cells were stably cotransfected with the Gi-coupled receptor for interleukin-8, CXCR1, and with a chimeric Gα, Gαqz5, which resembles Gαi in susceptibility to activation by Gi-coupled receptors but cannot regulate the Gαi effector, adenylyl cyclase. These cells, unlike cells expressing CXCR1 alone, migrated toward interleukin-8 even after treatment with pertussis toxin, which prevents activation of endogenous Gαi but not that of Gαqz5. We infer that chemotaxis does not require activation of Gαi. Because chemotaxis is mediated by Gβγ subunits released when Gi-coupled receptors activate Gαqz5, but not when Gq- or Gs-coupled receptors activate their respective G proteins, we propose that Gi-coupled receptors transmit a necessary chemotactic signal that is independent of Gαi.

C5a receptor activation. Genetic identification of critical residues in four transmembrane helices.

Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins). To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis. A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions. Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J. M., Schertler, G. F., and Unger, V. M. (1997) J. Mol. Biol. 272, 144–164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions. Our analysis identified 25 amino acid positions resistant to nonconservative substitutions. These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle. Twenty-one of the 121 mutant receptors exhibit constitutive activity. Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI. These results identify key elements of a general mechanism for the serpentine receptor switch.

Pseudohypoparathyroidism, a Novel Mutation in the βγ-Contact Region of Gsα Impairs Receptor Stimulation

Pseudohypoparathyroidism, type Ia (PHP-Ia), is a dominantly inherited endocrine disorder characterized by resistance to hormones that act by stimulating adenylyl cyclase. It is caused by inheritance of an autosomal mutation that inactivates the α subunit (αs) of Gs, the stimulatory regulator of adenylyl cyclase. In three members of a family, the PHP-Ia phenotype is associated with a mutation (R231H) that substitutes histidine for an arginine at position 231 in αs. We assessed signaling function of αs-WT versus αs-R231H transiently transfected in HEK293 cells. Hormone receptor-dependent stimulation of cAMP accumulation in cells expressing αs-R231H is reduced by ∼75% in comparison to cAMP accumulation in cells expressing αs-WT. A second mutation, αs-R201C, inhibits the GTPase turnoff reaction of αs, thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, αs-R231H/R201C, stimulates cAMP accumulation almost as well (∼80%) as does αs-R201C itself, indicating that the R231H mutation selectively impairs receptor-dependent signaling. In three-dimensional structures of G protein heterotrimers, Arg-231 is located in a region, switch 2, that is thought to interact with the βγ subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with βγ) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.

Second Site Suppressor Mutations of a GTPase-deficient G-Protein α-Subunit SELECTIVE INHIBITION OF Gβγ-MEDIATED SIGNALING

G proteins transmit signals from cell surface receptors to intracellular effectors. The intensity of the signal is governed by the rates of GTP binding (leading to subunit dissociation) and hydrolysis. Mutants that cannot hydrolyze GTP (e.g.GsαQ227L, Gi2αQ205L) are constitutively activated and can lead to cell transformation and cancer. Here we have used a genetic screen to identify intragenic suppressors of a GTPase-deficient form of the Gα in yeast, Gpa1Q323L. Sequencing revealed second-site mutations in three conserved residues, K54E, R327S, and L353Δ (codon deletion). Each mutation alone results in a complete loss of the βγ-mediated mating response in yeast, indicating a dominant-negative mode of inhibition. Likewise, the corresponding mutations in a mammalian Gi2α (K46E, R209S, L235Δ) lead to inhibition of Gβγ-mediated mitogen-activated protein (MAP) kinase phosphorylation in cultured cells. The most potent of these βγ inhibitors (R209S) has no effect on Gi2α-mediated regulation of adenylyl cyclase. Despite its impaired ability to release βγ, purified recombinant Gpa1R327S is fully competent to bind and hydrolyze GTP. These mutants will be useful for uncoupling Gβγ- and Gα-mediated signaling events in whole cells and animals. In addition, they serve as a model for drugs that could directly inhibit G protein activity and cell transformation.

Role of the cAMP Signaling Pathway in the Regulation of Gonadotropin-Releasing Hormone Secretion in GT1 Cells

We studied the signaling pathways coupling gonadotropin-releasing hormone (GnRH) secretion to elevations in cAMP levels in the GT1 GnRH-secreting neuronal cell line. We hypothesized that increased cAMP could be acting directly by means of cyclic nucleotide-gated (CNG) cation channels or indirectly by means of activation of cAMP-dependent protein kinase (PKA). We showed that GT1 cells express the three CNG subunits present in olfactory neurons (CNG2, -4.3, and -5) and exhibit functional cAMP-gated cation channels. Activation of PKA does not appear to be necessary for the stimulation of GnRH release by increased levels of cAMP. In fact, pharmacological inhibition of PKA activity caused an increase in the basal secretion of GnRH. Consistent with this observation activation PKA inhibited adenylyl cyclase activity, presumably by inhibiting adenylyl cyclase V expressed in the cells. Therefore, the stimulation of GnRH release by elevations in cAMP appears to be the result of depolarization of the neurons initiated by increased cation conductance by cAMP-gated cation channels. Activation of PKA may constitute a negative-feedback mechanisms for lowering cAMP levels. We hypothesize that these mechanisms could result in oscillations in cAMP levels, providing a biochemical basis for timing the pulsatile release of GnRH.