Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes

The subcellular localization of G_sα, G_iα1&2, G_iα3, and Gβ was studied in primary-cultured undifferentiated and differentiated, lipid replete, adipose cells. The results show a distinct distribution for each of these G-proteins and differences between differentiated and undifferentiated cells. All the G-proteins examined had a cytoplasmic localization; only G_iα1 and 2 showed a significant colocalization with the plasma membrane and this only in differentiated cells. Most studies using cells in culture have reported an intracellular localization for G-proteins, whereas in tissue sections the localization has been reported to be largely with the plasma membrane, with some intracellular localization. The results suggest that the cell-cell interactions or the specific geometry imposed by culture conditions favor the intracellular compared to peripheral localization of G-proteins. Alternately, the posttranslational modifications necessary for G-protein insertion in the plasma membrane may be deficient in cultured cells. J. Cell. Biochem. 65:259–266. © 1997 Wiley-Liss, Inc.     

Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Increases Functional Coupling Between a G Protein (GS) and Adenylyl Cyclase

Abstract: It has been reported that antidepressant treatment in rats results in a significant increase of G_s-mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down-regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl-5′-imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21-day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant-induced β-receptor desensitization occurred earlier than the enhancement of Gpp(NH)p-activated adenylyl cyclase, and extensive desensitization of β receptors by isoproterenol treatment did not enhance the Gpp(NH)p-stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p-stimulated adenylyl cyclase activity is not correlated with desensitization of β-adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially G_s) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (α_s, α_i, α_o, and β) nor their association with the plasma membrane was changed after antidepressant treatment. Thus, these results are consistent with the hypothesis that chronic antidepressant treatment acts directly at the postsynaptic membrane to increase the coupling between G_s and adenylyl cyclase.     

Modulation of adenylyl cyclase by FPP and adenosine involves stimulatory and inhibitory adenosine receptors and g proteins

FPP and adenosine modulate the adenylyl cyclase (AC)/cAMP signal transduction pathway in mammalian spermatozoa to elicit a biphasic response, initially stimulating capacitation and then inhibiting spontaneous acrosome loss. This study addressed the hypothesis that responses to FPP involve interactions between receptors for FPP and adenosine, the biphasic responses involving stimulatory and inhibitory adenosine receptors. Gln‐FPP, a competitive inhibitor of FPP, significantly inhibited binding of an adenosine analogue and responses to adenosine, especially in capacitated suspensions, consistent with interaction between FPP and adenosine receptors. CGS‐21680 (1 μM), a stimulatory A_2a adenosine receptor agonist, significantly stimulated capacitation and cAMP in uncapacitated cells, while cyclopentyl adenosine (1 μM), an inhibitory A₁ adenosine receptor agonist only affected capacitated cells, inhibiting spontaneous acrosome loss. Responses to FPP and adenosine were inhibited in uncapacitated cells by a selective A_2a antagonist and in capacitated cells by a selective A₁ antagonist; subsequent investigations indicated possible involvement of G proteins. Like FPP, cholera toxin stimulated capacitation and cAMP production in uncapacitated cells, suggesting involvement of a G protein with a Gα_s subunit. In contrast, pertussis toxin prevented FPP's inhibition of both spontaneous acrosome loss and cAMP production, suggesting involvement of a Gα_i/_o subunit. Immunoblotting evidence revealed the presence of proteins of the appropriate molecular weights for Gα_s, Gα_i2, Gα _i3, and Gα_o subunits. This study provides the first direct evidence suggesting the involvement of two different types of adenosine receptors and both Gα_s and Gα_i/_o subunits in the regulation of capacitation, resulting in modulation of AC activity and availability of cAMP. Mol. Reprod. Dev. 53:459–471, 1999. © 1999 Wiley‐Liss, Inc.     

β3-Adrenergic activation of adenylyl cyclase in mouse white adipocytes: modulation by GTP and effect of obesity

Lipolysis and adenylyl cyclase (AC) activation in response to β-adrenergic agents are abnormally low in white epididymal adipose tissue (WAT) of the ob/ob mouse. The abundance of G-proteins (G_sα and G_iα) linked to AC is also abnormally low. By contrast, β-adrenergic receptor (β-AR) levels were previously found to be normal in WAT and elevated in liver. The relative importance of various forms of the β-AR in mouse WAT was reassessed in view of the discovery of the β₃-AR. The results show that (1) the β₃-AR is mainly responsible for AC activation in lean-mouse WAT; (2) the β₃-AR is only partly responsible for AC activation in obese mouse WAT; and (3) GTP modulates β₃—-but not β₁—-or β₂-AR activation of AC in a biphasic manner. Therefore, the β₃-AR appears responsible for the well-known bimodal effect of GTP on β-adrenergic receptor-mediated AC activity in WAT.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.     

Retinoic Acid-Induced Differentiation of Human Neuroblastoma SH-SY5Y Cells Is Associated with Changes in the Abundance of G Proteins

Abstract: Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH- SY5Y cells: Gaα, G_iα1, G_jα2, G_cα, G_zα, and Gβ. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 μmol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of μ-opioid binding sites, the levels of the inhibitory G proteins G_iα1 and G_jα1 were found to be significantly increased. This coordinate up-reg- ulation is accompanied by functional changes in μ-opioid receptor-stimulated Iow-K_m GTPase, μ-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5′-(βγ-imido)triphosphate [Gpp(NH)p; 10 nmol/ L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prosta- glandin E₁ (PGE₁) receptors and G_sα, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE₁ binding sites and PGE₁ stimulated adenylate cyclase activity, but significantly reduced amounts of G_zα were found. This down- regulation is paralleled by a decrease in the stimulatory activity of G_zα as assessed in S49 cyc- reconstitution assays. However, the reduction in G_aα levels had no effect on both intrinsic and receptor-independent-activated [Gpp(NH)p or forskolin; 100 μtmol/L each] adenylate cyclase, suggesting that the amount of G_zα is in excess over the functional capacity of adenylate cyclase in SH-SY5Y cell membranes. Additional quantitative changes were found for G_zα, G_cα, and Gβ subunits. In contrast, neuronal differentiation in the presence of 12-O-tetradecanoylphor- bol 13-acetate (16 nmol/L; 6 days) failed to affect G protein abundance. Our results provide evidence for a specific RA effect on the abundance of distinct G protein sub- units in human SH-SY5Y neuroblastoma cells. These alterations might contribute to functional changes in transmembrane signaling pathways associated with RA-in- duced neuronal differentiation of the cells.     

Pertussis-toxin insensitive enkephalin inhibition of adenylyl cyclase in lacrimal acinar cells

The mechanism by which the inhibitory effect of d-ala²-met-enkephalinamide (DALA) on lacrimal acinar adenylyl cyclase is exerted was assessed in membrane preparations by a cAMP protein binding assay. Inhibition by the analogue was GTP-dependent with a significant enhancement of the inhibitory effect by GTP. While pretreatment of membranes with either cholera or pertussis toxin resulted in stimulation of adenylyl cyclase activity, modification of the G_iα subunit by pertussis-toxin catalyzed ADP-ribosylation did not effect the hormonal inhibition of adenylyl cyclase. Incubation of membranes with manganese, however, prevented the inhibitory action of DALA in addition to enhancing basal and forskolin-stimulated adenylyl cyclase activity. The results suggest that the inhibitory effect of DALA in lacrimal acinar cells is exerted via a mechanism other than pertussis-toxin sensitive coupling of the receptor to adenylyl cyclase through G_i. The mechanism may be effected through a pertussis-toxin insensitive G protein, through an interaction with G_i that is pertussis-toxin insensitive, or through an interaction with the catalytic subunit of adenylyl cyclase.     

The effects of the deep-sea environment on transmembrane signaling

Membrane-associated processes may be particularly susceptible to perturbation by the high hydrostatic pressures and low temperatures of the deep ocean. Transmembrane signaling by guanyl nucleotide binding protein (G protein) coupled receptors (GPCRs) is affected at a number of steps: (1) agonist activation of the GPCR; (2) the interaction of the receptor with the heterotrimeric G protein; (3) the G protein GTPase cycle; and (4) the activation and function of the effector element, adenylyl cyclase. The effects of low temperature and high hydrostatic pressures on the A₁ adenosine receptor–inhibitory G protein (G_i)–adenylyl cyclase signaling complex were examined in teleost fishes from three families, Scorpaenidae, Macrouridae and Moridae. In a comparison of teleost fishes, rat and chicken, species with body temperatures from 1 to 40 °C, at atmospheric pressure, A₁ adenosine receptor agonist binding is conserved at the body temperature of the species. In the marine teleost fishes examined, increased pressure decreases agonist efficacy. There are differences among species in the effects of increased hydrostatic pressure on G protein interactions with receptors, GTP binding to G protein α subunits and the intrinsic GTPase activity of α subunits. Adenylyl cyclase activity and modulation are affected by increased pressure in all the species examined, except Antimora rostrata which was unaffected by pressure changes. At pressures approximating those which the species experience in situ adenylyl cyclase activity retains its sensitivity to modulators. To understand the physiological consequences of impaired cell signaling several prototypical human diseases are discussed.     

Structure,Function, and Regulation of the Three β-Adrenergic Receptors

Three β-adrenergic receptor subtypes are now known to be functionally expressed in mammals. All three belong to the R₇G family of receptors coupled to G-proteins, and characterized by an extracellular glycosylated N-terminal and an intracellular C-terminal region and seven transmembrane domains, linked by three exta- and three intracellular loops. The catecholamine ligand binding domain, studied using affinity-labeling and site-directed mutagenesis, is a pocket lined by residues belonging to the transmembrane domains. The region responsible for the interaction with the Gs protein which, when activated, stimulates adenylyl cyclase, is composed of residues belonging to the parts most proximal to the membrane of intracellular loop i₃ and the C-terminal region. The pharmacology of the three subtypes is quite distinct: in fact most of the potent β₁/β₂ antagonists (the well known β blockers) act as agonists on β₃. The subtype is resistant to short-term desensitization mediated by phosphorylation through PKA or βARK, in stark contrast to the β₁ or β₂ subtypes. Various compounds (dexamethasone, butyrate, insulin) up regulate β₁ or β₁ subtypes while down-regulating β₃ whose expression strictly correlates with differentiation of 3T3-F442A fibroblasts into adipocytes, thus confirming that the expression of the three subtypes may each be regulated independently to exert a specific physiologic role in different tissues or at different stages of development.     

Trp fluorescence reveals an activation‐dependent cation‐π interaction in the Switch II region of Gαi proteins

Crystal structures of Gα_i (and closely related family member Gα_t) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Gα_t exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP‐bound state, unlike Gα_i, which exhibits higher basal exchange and a disordered Switch II region in Gα_iGDP structures. Using purified Gα_i and Gα_t, we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Gα proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GαGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation‐π interaction between tryptophan and arginine residues in the Switch II of Gα_i family proteins that mediates the observed red shift in emission maxima. Furthermore, amino‐terminal myristoylation of Gα_i resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Gα proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.     

Human-Xenopus chimeras of Gsα reveal a new region important for its activation of adenylyl cyclase

G proteins are heterotrimeric GTPases that play a key role in signal transduction. The α subunit of G_s bound to GTP is capable of activating adenylyl cyclase. The amino acid sequences derived from two X. laevis cDNA clones that apparently code for G_sα subunits are 92% identical to those found in the short form of human G_sα. Despite this high homology, the X. laevis G_sα clones expressed in vitro, yielded a protein that are not able to activate the adenylyl cyclase present in S49 cyc⁻ membranes in contrast with human G_sα similarly expressed. This finding suggested that the few amino acid substitutions found in the amphibian subunit are important in defining the functionality of the human G_sα. The construction of chimeras composed of different fractions of the cDNAs of the two species was adopted as an approach in determining the regions of the molecule important in its functionality in this assay. Four pairs of chimeras were constructed using reciprocal combinations of the cDNAs coding for human and Xenopus G_sα. These eight constructs were expressed in vitro and equivalent amounts of the resulting proteins were assayed in the activation of adenylyl cyclase with GTPγs and isoproterenol. The results obtained here clearly indicate that the Gα sequence that extends from amino acid 70 to 140, is important for the functionality of human G_sα in activating adenylyl cyclase.     

Phosphatidylinositol‐3‐OH kinase regulatory subunits are differentially expressed during development of the rat cerebellum

Recent evidence implicates a central role for PI3K signalling in mediating cell survival during the process of neuronal differentiation. Although PI3K activity is stimulated by a wide range of growth factors and cytokines in different cell lines and tissues, activation of this pathway by insulin‐like growth factor I (IGF‐I) most likely represents the main survival signal during neuronal differentiation. IGF‐I is highly expressed during development of the central nervous system, and thus is a critical factor for the development and maturation of the cerebellum. Upon ligand binding, the IGF‐I receptor phosphorylates tyrosine residues in SHC and insulin receptor substrates (IRSs) initiating two main signalling cascades, the MAP kinase and the phosphatidylinositol 3‐kinase (PI3K) pathways. Activated PI3K is composed of a catalytic subunit (p110α or β) associated with one of a large family of regulatory subunits (p85α, p85β, p55γ, p55α, and p50α). To evaluate the contributions of these various regulatory subunits to neuronal differentiation, we have used antibodies specific for each of the PI3K subunits. Using these antisera, we now demonstrate that PI3K subunits are differentially regulated in cerebellar development, and that the expression level of the p55γ regulatory subunit reaches a maximum during postnatal development, decreasing thereafter to low levels in the adult cerebellum. Furthermore, our studies reveal that the distribution of the various PI3K regulatory subunits varies during development of the cerebellum. Interestingly, p55γ is expressed in both glial and neuronal cells; moreover, in Purkinje neurones, this subunit colocalises with the IGF‐IR. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 39–50, 2001     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

Effect of activating and inactivating mutations of GS-and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes

Previous investigations have demonstrated that both G_s- and the G_i-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of G_sα vs. G_i2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-G_sα or the inactivating G226A(H21a)-G_sα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G_i2α wild type or the missense mutations R179E-G_i2α, Q205L-G_i2α, and G204A(H21a)-G_i2α. The activating [R201C]G_sα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]G_sα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]G_i2α or [Q205L]G_i2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]G_i2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]G_i2α or [Q205L]G_i2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G_i2α and [Q205L]G_i2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]G_i2α and [Q205L]G_i2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G_i2α and [Q205L]G_i2α over differentiated controls. The inactivating [H21a]G_i2α-mutant obliterated all signs of preadipocyte differentiation. It is concluded that G_i2 plays a positive and much more important role than G_s in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242–257. © 1997 Wiley-Liss, Inc.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins

The function of guanine nucleotide binding (G) proteins is Mg²⁺ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg²⁺ binding sites are present or if one Mg²⁺ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg²⁺-specific fluorophore, to investigate Mg²⁺ binding to α subunits in both conformations of the stimulatory (G_sα) and inhibitory (G_iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg²⁺ sites were present with dissimilar affinities. With the exception of G_sα in the active conformation, cooperativity between the two Mg²⁺ sites was also observed. Whereas the high affinity Mg²⁺ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg²⁺ site may involve coordination to the terminal phosphate of the nucleotide.     

多巴胺受体的cDNA克隆

通过多巴胺受体的5个cDNA克隆,综述和分析了5个多巴胺受体(D₁R-D₅R)的基因结构,在染色体上的定位及其mRNA在中枢脑区的分布;比较了这5个受体cDNA克隆的结构特征和药理学性质.