Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.

The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.     

GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP- dependent reaction during protein translocation is the SRP receptor- mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.     

Structural basis for the function of the beta subunit of the eukaryotic signal recognition particle receptor

Protein translocation across and insertion into membranes is a process essential to all life forms. In higher eukaryotes, this process is initiated by targeting the translating ribosome to the endoplasmic reticulum via the signal recognition particle (SRP) and its membrane-associated heterodimeric receptor (SR). This targeting step is regulated by three G proteins, SRP54, SR alpha, and SR beta, which act in concert. Little is known about the regulatory role of SR beta. Here, we present the 1.7 A crystal structure of the SR beta-GTP subunit in complex with the interaction domain of SR alpha. Strikingly, the binding interface overlaps largely with the switch 1 region of SR beta. This finding, together with additional biochemical data, shows that the eukaryotic SR is a conditional and not an obligate heterodimer. The results suggest that the GTP/GDP switch cycle of SR beta functions as a regulatory switch for the receptor dimerization. We discuss the implications for the translocation pathway.     

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.     

The conformation of bound GMPPNP suggests a mechanism for gating the active site of the SRP GTPase

BACKGROUND: The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that mediates cotranslational targeting of secreted and membrane proteins to the membrane. Targeting is regulated by GTP binding and hydrolysis events that require direct interaction between structurally homologous "NG" GTPase domains of the SRP signal recognition subunit and its membrane-associated receptor, SR alpha. Structures of both the apo and GDP bound NG domains of the prokaryotic SRP54 homolog, Ffh, and the prokaryotic receptor homolog, FtsY, have been determined. The structural basis for the GTP-dependent interaction between the two proteins, however, remains unknown. RESULTS: We report here two structures of the NG GTPase of Ffh from Thermus aquaticus bound to the nonhydrolyzable GTP analog GMPPNP. Both structures reveal an unexpected binding mode in which the beta-phosphate is kinked away from the binding site and magnesium is not bound. Binding of the GTP analog in the canonical conformation found in other GTPase structures is precluded by constriction of the phosphate binding P loop. The structural difference between the Ffh complex and other GTPases suggests a specific conformational change that must accompany movement of the nucleotide from an "inactive" to an "active" binding mode. CONCLUSIONS: Conserved side chains of the GTPase sequence motifs unique to the SRP subfamily may function to gate formation of the active GTP bound conformation. Exposed hydrophobic residues provide an interaction surface that may allow regulation of the GTP binding conformation, and thus activation of the GTPase, during the association of SRP with its receptor.     

SRbeta coordinates signal sequence release from SRP with ribosome binding to the translocon

Protein targeting to the endoplasmic reticulum (ER) membrane is regulated by three GTPases, the 54 kDa subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). Using a soluble form of SR and an XTP-binding mutant of SRbeta, we show that SRbeta is essential for protein translocation across the ER membrane. SRbeta can be cross-linked to a 21 kDa ribosomal protein in its empty and GDP-bound state, but not when GTP is bound. GTP binding to SRbeta is required to induce signal sequence release from SRP. This is achieved by the presence of the translocon, which changes the interaction between the 21 kDa ribosomal protein and SRbeta and thereby allows SRbeta to bind GTP. We conclude that SRbeta coordinates the release of the signal sequence from SRP with the presence of the translocon.     

Crystal structure of SRP19 in complex with the S domain of SRP RNA and its implication for the assembly of the signal recognition particle

The signal recognition particle (SRP) is a ribonucleoprotein particle involved in GTP-dependent translocation of secretory proteins across membranes. In Archaea and Eukarya, SRP19 binds to 7SL RNA and promotes the incorporation of SRP54, which contains the binding sites for GTP, the signal peptide, and the membrane-bound SRP receptor. We have determined the crystal structure of Methanococcus jannaschii SRP19 bound to the S domain of human 7SL RNA at 2.9 A resolution. SRP19 clamps the tetraloops of two branched helices (helices 6 and 8) and allows them to interact side by side. Helix 6 acts as a splint for helix 8 and partially preorganizes the binding site for SRP54 in helix 8, thereby facilitating the binding of SRP54 in assembly.     

Structure of the chloroplast signal recognition particle (SRP) receptor: domain arrangement modulates SRP-receptor interaction

The signal recognition particle (SRP) pathway mediates co-translational targeting of nascent proteins to membranes. Chloroplast SRP is unique in that it does not contain the otherwise universally conserved SRP RNA, which accelerates the association between the SRP guanosine-5′-triphosphate (GTP) binding protein and its receptor FtsY in classical SRP pathways. Recently, we showed that the SRP and SRP receptor (SR) GTPases from chloroplast (cpSRP54 and cpFtsY, respectively) can interact with one another 400-fold more efficiently than their bacterial homologues, thus providing an explanation as to why this novel chloroplast SRP pathway bypasses the requirement for the SRP RNA. Here we report the crystal structure of cpFtsY from Arabidopsis thaliana at 2.0 Å resolution. In this chloroplast SR, the N-terminal “N” domain is more tightly packed, and a more extensive interaction surface is formed between the GTPase “G” domain and the N domain than was previously observed in many of its bacterial homologues. As a result, the overall conformation of apo-cpFtsY is closer to that found in the bacterial SRP•FtsY complex than in free bacterial FtsY, especially with regard to the relative orientation of the N and G domains. In contrast, active-site residues in the G domain are mispositioned, explaining the low basal GTP binding and hydrolysis activity of free cpFtsY. This structure emphasizes proper N-G domain arrangement as a key factor in modulating the efficiency of SRP-receptor interaction and helps account, in part, for the faster kinetics at which the chloroplast SR interacts with its binding partner in the absence of an SRP RNA.     

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

The ribosome regulates the GTPase of the beta-subunit of the signal recognition particle receptor.

Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the alpha- and beta-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the beta-subunits of the SR (SRbeta). We found that SRbeta binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRbeta and thus functions as a GTPase activating protein for SRbeta. Furthermore, the interaction between SRbeta and the ribosome leads to a reduction in the affinity of SRbeta for guanine nucleotides. We propose that SRbeta regulates the interaction of SR with the ribosome and thereby allows SRalpha to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SRalpha then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.     

A novel guanine nucleotide-binding protein coupled to the alpha 1-adrenergic receptor. II. Purification, characterization, and reconstitution

In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.     

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.     

Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human autoantibodies against the 54 kDa protein of the signal recognition particle block function at multiple stages

The 54 kDa subunit of the signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and membrane proteins and it contributes to the targeting of these precursors to the membrane of the endoplasmic reticulum (ER). At the ER membrane, the binding of the signal recognition particle (SRP) to its receptor triggers the release of SRP54 from its bound signal sequence and the nascent polypeptide is transferred to the Sec61 translocon for insertion into, or translocation across, the ER membrane. In the current article, we have characterized the specificity of anti-SRP54 autoantibodies, which are highly characteristic of polymyositis patients, and investigated the effect of these autoantibodies on the SRP function in vitro. We found that the anti-SRP54 autoantibodies had a pronounced and specific inhibitory effect upon the translocation of the secretory protein preprolactin when analysed using a cell-free system. Our mapping studies showed that the anti-SRP54 autoantibodies bind to the amino-terminal SRP54 N-domain and to the central SRP54 G-domain, but do not bind to the carboxy-terminal M-domain that is known to bind ER signal sequences. Nevertheless, anti-SRP54 autoantibodies interfere with signal-sequence binding to SRP54, most probably by steric hindrance. When the effect of anti-SRP autoantibodies on protein targeting the ER membrane was further investigated, we found that the autoantibodies prevent the SRP receptor-mediated release of ER signal sequences from the SRP54 subunit. This observation supports a model where the binding of the homologous GTPase domains of SRP54 and the α-subunit of the SRP receptor to each other regulates the release of ER signal sequences from the SRP54 M-domain.     

Identification and characterization of Streptococcus pneumoniae Ffh, a homologue of SRP54 subunit of mammalian signal recognition particle

Recent studies have demonstrated that bacteria possess an essential protein translocation system similar to mammalian signal recognition particle (SRP). Here we have identified the Ffh, a homologue of the mammalian SRP54 subunit from S. pneumoniae. Ffh is a 58-kDa protein with three distinct domains: an N-terminal hydrophilic domain (N-domain), a G-domain containing GTP/GDP binding motifs, and a C-terminal methionine-rich domain (M-domain). The full-length Ffh and a truncated protein containing N and G domains (Ffh-NG) were overexpressed in E. coli and purified to homogeneity. The full-length Ffh has an intrinsic GTPase activity with k(cat) of 0.144 min(-1), and the K(m) for GTP is 10.9 microM. It is able to bind to 4.5S RNA specifically as demonstrated by gel retardation assay. The truncated Ffh-NG has approximately the same intrinsic GTPase activity to the full-length Ffh, but is unable to bind to 4.5S RNA, indicating that the NG domain is sufficient for supporting intrinsic GTP hydrolysis, and that the M domain is required for RNA binding. The interaction of S. pneumoniae Ffh with its receptor, FtsY, resulted in a 20-fold stimulation in GTP hydrolysis. The stimulation was further demonstrated to be independent of the 4.5S RNA. In addition, a similar GTPase stimulation is also observed between Ffh-NG and FtsY, suggesting that the NG domain is sufficient and the M domain is not required for GTPase stimulation between Ffh and FtsY.     

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP-receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP-receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP-receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo.     

Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.     

The alpha 2B adrenergic receptor of undifferentiated neuroblastoma x glioma hybrid NG108-15 cells, interacts directly with the guanine nucleotide binding protein, Gi2

In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2.