Two forms of the bovine brain Go that stimulate the inositol trisphosphate-mediated Cl- currents in Xenopus oocytes. Distinct guanine nucleotide binding properties.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.

Distinct guanine nucleotide binding and release properties of the three Gi proteins.

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.

Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system.

The selectivity of D2 dopamine receptor-guanine nucleotide-binding protein (G protein) coupling was studied by reconstitution techniques utilizing purified D2 dopamine receptors from bovine anterior pituitary and resolved G proteins from bovine brain, bovine pituitary, and human erythrocyte. Titration of a fixed receptor concentration with varying G protein concentrations revealed two aspects of receptor-G protein coupling. First, Gi2 appeared to couple selectively with the D2 receptor with approximately 10-fold higher affinity than any other tested Gi subtype. Second, the G proteins differed in the maximal receptor-mediated agonist stimulation of the intrinsic GTPase activity. Gi2 appeared to be maximally stimulated by agonist-receptor complex with turnover numbers of approximately 2 min-1. The other Gi subtypes, Gi1 and Gi3, could be only partially activated, resulting in maximal rates of GTPase of approximately 1 min-1. Agonist-stimulated GTPase activity was not detected in preparations containing Go from bovine brain. The differences in maximal agonist-stimulated GTPase rates observed among the Gi subtypes could be explained by differences in agonist-promoted guanyl nucleotide exchange. Both guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding and GDP release parameters were enhanced 2-fold for the Gi2 subtype over the other Gi subtypes. These results suggest that even though several types of pertussis toxin substrate may exist in most tissues, a receptor may interact discretely with G proteins, thereby dictating signal transduction mechanisms.

Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway.

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, we have used the Xenopus oocyte in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. We report here that the activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. We also show that holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes.

The G protein-gated atrial K+ channel is stimulated by three distinct Gi alpha-subunits.

The guanine nucleotide-binding protein, Gi, which inhibits adenylyl cyclase, has recently been shown to have three subtypes of the alpha-subunit, termed Gi alpha-1, Gi alpha-2 and Gi alpha-3. They share 87-94% amino-acid sequence homology and so are difficult to separate from one another. Among other functions, purified preparations activate K+ channels but there is confusion over which of the subtypes activates the muscarinic K+ channels of the atrial muscle of the heart: Gi alpha-3, also termed Gk, has been shown to activate this channel but it is not clear whether Gi alpha-1 does or does not. To clarify this problem, we expressed the subtypes separately in Escherichia coli to eliminate contamination by other subtypes and tested the recombinant alpha- chains on atrial muscarinic K+ channels. Although we anticipated that only Gi alpha-3 would have Gk activity, to our surprise all three recombinant subtypes were active, from which we deduce that the Gi subtypes are multifunctional.

The hepatic glucagon receptor: a comparative study of the regulatory and structural properties.

Guanine nucleotide and Mg2+ ion regulation of [125I-Tyr10]monoiodoglucagon ([125I]MIG) binding to liver plasma membranes from chicken, rat, and rabbit was studied. It was found that [125I]MIG binding to chicken liver membranes was increased by the addition of Mg2+ ion, while binding to rat and rabbit liver membranes was unaffected. In the chicken liver membranes, the Mg2+ ion induced high affinity binding which was sensitive to guanine nucleotides, while the low affinity binding in the absence of Mg2+ ion was not. Maximal effects of Mg2+ ion were observed at 1 mM. Glucagon binding to rat liver membrane receptors was GTP sensitive regardless of whether Mg2+ ion was added. Glucagon binding to rabbit liver membranes was insensitive to both Mg2+ ions and GTP. This lack of GTP effect was not due to degradation of GTP; no effect of the nonhydrolyzable analog guanyl-5'-yl-imidodiphosphate was observable. Glucagon stimulation of rabbit liver adenylyl cyclase, however, was dependent on GTP, as was the case with all of the other liver adenylyl cyclases studied here. The Kact of GTP for the rabbit liver system was very similar to that for rat liver membranes. The glucagon receptor was covalently labeled with [125I]MIG using p-hydroxysuccinimidyl azidobenzoate and analyzed by sodium dodecyl sulfate-gel electrophoresis. In all cases, a major labeled band at 63,000 daltons was observed. The levels of glucagon receptor and stimulatory (Ns) and inhibitory (Ni) regulatory proteins of adenylyl cyclase were measured. The highest levels of glucagon receptor were measured in rat liver membranes, while the levels in chicken and rabbit membranes were 30-40% lower. Rabbit liver membrane had the highest levels of Ns, while rat liver membranes had 2-fold lower and chick liver membrane 4-fold lower levels than rabbit liver membranes. The levels of Ni was similar in the three systems. Thus, the ratio of Ns to glucagon receptor was highest in the rabbit. In the rat, this ratio was 3-fold lower than that in the rabbit. In the chicken membranes, the ratio was about 60% of that in the rat. These data suggest that the observed differences in effects of GTP on hormone binding can be explained by alterations in the ratio of the receptor and Ns proteins among the various species.