Introduction

Summary Introduction Procedures Results Discussion References

A host of clinically important drug receptors use heterotrimeric () G proteins belonging to the G_i subfamily for signal transduction. The G_i-coupled receptors include those that bind catecholamines, acetylcholine, serotonin, histamine, opioids, chemoattractants, chemokines, and many neuropeptides. Molecular determinants conferring selectivity at the receptor/G protein interface are beginning to be mapped out (1). Ample evidence is available in support of the notion that the carboxyl terminus of the  subunit of G_i proteins is one of the major contact sites with receptors. Attachment of an ADP-ribose moiety to the carboxyl tail of _i by PTX prevents receptor-induced G_i activation (2). Antisera against the extreme carboxyl termini of the _i chains can effectively disrupt receptor-induced inhibition of AC (3). Moreover, replacement of the last five amino acids of q with the i2 carboxyl end sequence allows the chimeric q/i2 protein to transduce signals from receptors that are normally coupled to G_i proteins (4). The importance of the carboxyl terminus in receptor recognition is also supported by studies with other  chains. An unc mutation at residue 389 uncouples s from the -adrenoceptor (5), whereas synthetic peptides derived from the carboxyl terminus of t1 (rod transducin) have been shown to curtail rhodopsin/t1 interaction (6).

Although less apparent, the amino terminus may also contribute toward the binding of i subunits to receptors. Inferences can be drawn from the observations that the mastoparan-induced activation of G_o is abolished by point mutations (7) or tryptic digestion (8) of the amino terminus of o. Synthetic peptides derived from the amino terminus of t1 have been shown to inhibit effective interactions between rhodopsin and t1 (6, 9). More recently, analysis of the crystal structures of i1 (10) and i112 (11) have suggested that both amino and carboxyl termini may be involved in the coupling of the  chain to the receptor. However, because the  chain amino terminus is also involved in binding to the complex (11, 12), its contribution to receptor coupling may be indirect.

The cysteine residue at the carboxyl termini of i chains, which can be ADP-ribosylated by PTX, does not seem to be required for receptor coupling. For instance, substitution of this cysteine in o with serine removes the PTX-induced inhibition but does not affect the transduction of hormonal signals via G_o (13). A similar mutation on i2 produced a functional i2 chain that acquired resistance to PTX (14). Perhaps the most convincing evidence comes from the demonstration that G_z, a PTX-insensitive G protein (15, 16), can mediate hormonal inhibition of AC by interacting with a wide variety of G_i-coupled receptors (17-22). Despite the fact that i and z share only ~60% identity in their amino acid sequences, their functional similarity suggests that they may share similar domains for receptor coupling. As a first step toward identifying the critical molecular determinants for receptor recognition by G_i proteins, we constructed a series of chimeric  subunits by swapping the carboxyl termini of i2 and z with each other or with those of other  subunits, such as t1, 12, and 13. Their functional coupling to receptors was then assessed by measuring -mediated stimulation of AC II in human embryonic kidney 293 cells transiently coexpressing the various chimeras.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Materials. The human FMLP receptor and the murine -opioid receptor cDNAs (both in the pCDM8 vector) were generous gifts from Dr. F. Boulay (Laboratoire de Biochemie, Grenoble, France) and Dr. C. Evans (University of California, Los Angeles), respectively. The rat µ-opioid receptor cDNA (in the pRc/CMV vector) and the mouse -opioid receptor (in the pCMV6 vector) were kindly provided by Dr. L. Yu (Indiana University School of Medicine, Indianapolis, IN) and Dr. G. Bell (University of Chicago, Chicago, IL), respectively. cDNAs encoding the  subunits of G₁₂ and G₁₃ were supplied by Dr. M. Simon (California Institute of Technology, Pasadena). Other cDNAs were constructed or obtained as previously described (17, 23). PTX was purchased from List Biological Laboratories (Campbell, CA). hCG was generously provided by the National Pituitary Agency (Bethesda, MD). Human embryonic kidney 293 cells were obtained from the American Type Culture Collection (CRL-1573, Rockville, MD). [³H]Adenine was purchased from Amersham (Arlington Heights, IL) Plasmid purification columns were obtained from Qiagen (Hilden, Germany). Antiserum 3A-170 against the carboxyl terminal of z was obtained from Gramsch Laboratories (Schwabhausen, Germany). Specific antisera for z (SC-388), 12 (SC-409), and 13 (SC-410) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD), and all other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Construction of chimeric  subunits. The i2/z chimera (in pcDNA1) was made by replacing a BglII/NotI fragment of mouse i2 with the corresponding sequence from rat z, so the last 36 residues of i2 were substituted with z sequence. z/i2 was created in a converse manner. The constitutively active i2/z-QL and z/i2-QL chimeras were constructed in a similar manner by using i2-Q205L (23) and z-Q205L (17) as the starting materials. To construct the t1/z chimera, t1 in pcDNA1 was digested with BglII and NdeI, and the 1.2-kb fragment was replaced with a 1.5-kb BglII/NdeI cognate fragment from z. The z/t1 chimera was made in a similar fashion, and the z/t1-QL chimera was made with z-Q205L as the source of z sequence. Construct identity was verified by restriction mapping. The cDNAs encoding the 12/z and 13/z chimeras were kindly provided by Henry Bourne (University of California, San Francisco). The source and construction of other cDNAs were previously reported (17, 23).

Cell culture and transfection. Human embryonic kidney 293 cells were maintained and transfected as previously reported (24). Briefly, cells were cultured in Eagle's minimum essential medium containing 10% (v/v) fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin in 5% CO₂ at 37°. Cells were seeded onto 12-well plates at ~1 × 10⁵ cells/well. One day later, cells were transfected with medium containing the desired cDNAs along with 400 µg/ml DEAE-Dextran and 0.1 mM chloroquine for 2 hr at 37°. The cells were then shocked with 10% (v/v) dimethylsulfoxide in phosphate-buffered saline and returned to growth medium. The efficiency of transfections was routinely monitored through coexpression of -galactosidase as a reporter.

cAMP accumulation. The transfected 293 cells were labeled 1 day later with [³H]adenine (1 µCi/ml) in minimum essential medium containing 1% (v/v) fetal calf serum. Where indicated, 100 ng/ml PTX was added simultaneously. After 16-20 hr, the cells were assayed for cAMP levels in response to various drugs as previously described (24). cAMP accumulations were determined in the presence of 1 mM 1-methyl-3-isobutylxanthine at 37° for 30 min. Results are expressed as the ratios of [³H]cAMP to total [³H]ATP, [³H]ADP, and [³H]cAMP pools. Absolute values for cAMP accumulation varied between experiments, but variability within a given experiment was <10% in general.

Immunodetection of chimeric  subunits. Membranes were prepared from transiently transfected 293 cells. Briefly, transfected cells were harvested in phosphate-buffered saline (Ca²⁺ and Mg²⁺ free) containing 10 mM EDTA. Cells were resuspended in lysis buffer (50 mM Tris·HCl containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine-HCl, 1 mM EGTA, 5 mM MgCl₂, and 1 mM dithiothreitol, pH 7.4) and lysed by one cycle of freeze-thawing followed by 10 passages through a 27-guage needle. After removal of nuclei by centrifugation, membranes were collected, washed, and resuspended in lysis buffer. Protein concentrations were determined using the BioRad (Hercules, CA) Protein Assay Kit. For each sample, 75 µg of membrane proteins was separated on a µ polyacrylamide-sodium dodecyl sulfate gel and electrophoretically transferred to polyvinylidene difluoride membranes. Localization of protein markers on the polyvinylidene difluoride membrane was by Ponceau S staining. The following antisera were used: z-specific 3A-170 and SC-388 for t1/z and z/t1, respectively; 12-specific SC-409 for 12/z; and 13-specific SC-410 for 13/z. Antigen/antibody complexes were visualized by enhanced chemiluminescence using the ECL kit from Amersham.

	Results

Summary Introduction Procedures Results Discussion References

Chimeras of i2 and z can interact with the -opioid receptor. The last 36 amino acids at the carboxyl termini of i2, z, and t chains share substantial identity (~70%); the overall charge distribution of this region varies between  chains due to differences in three to five amino acids. A conserved BglII restriction site allows the swapping of the carboxyl terminal ends of these  subunits with each other after digestion of their cDNAs with restriction endonuclease and religation of corresponding fragments. The carboxyl terminal region of i2 contains three charged residues (K331, K346, and D351) that are not found in the z sequence. To test whether the i2/z chimera can interact with typical G_i-coupled receptors, we made use of a recombinant assay system that uses the -mediated stimulation of AC II. This assay is based on the finding that in the presence of an activated s, many receptors have the ability to stimulate AC II activity by releasing the subunits from G_i and G_z proteins (20, 22, 25-27). We have thus cotransfected 293 cells with cDNAs encoding AC II, s-Q227L (a constitutively active mutant of s; Ref. 28), -opioid receptor, and one of several  subunits in their wild-type (i2 and z) or chimeric (i2/z and z/i2) forms. We have previously shown that under these experimental conditions, 293 cells exhibited stimulation of cAMP production in response to activation of G_i-coupled receptors (20, 22, 26, 27), indicating the expression of a functional AC II in this cell system.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Potentiation of DPDPE-induced stimulation of AC II by i2/z and z/i2 chimeras. The 293 cells were transiently cotransfected (DEAE-Dextran method), labeled with [3H]adenine (1 µCi/ml), and then assayed for cAMP accumulation in the absence or presence of 100 nM DPDPE as described in the text. Cotransfections were with AC II (0.25 µg/ml), s-Q227L (0.025 µg/ml), the -opioid receptor (0.25 µg/ml), and 0.15 µg/ml concentration of one of the following cDNAs: i2, z, i2/z, z/i2, or pcDNA1 vector (control). Cells were subjected to PTX treatment (100 ng/ml, 16 hr) where indicated. Data shown represent triplicate determinations in a single experiment; two independent experiments yielded similar results. Results are expressed as percent stimulation of cAMP formation in the presence of DPDPE compared with that measured in the absence of DPDPE. Basal values are expressed as the ratio (×103) of cAMP to total adenine nucleotides and ranged from 6.55 ± 0.52 to 9.24 ± 0.43. *, DPDPE-stimulated cAMP accumulation was significantly different from that observed in the vector-transfected cells (paired Bonferroni t test, p < 0.05). **, Significantly higher than basal cAMP accumulation in PTX-treated cells (paired t test, p < 0.05). Left, schematic of the various wild-type and chimeric  subunits used in the cotransfections.

Inhibition of AC by i2/z and z/i2. We have previously shown that both i2 and z inhibit cAMP accumulation to similar extents (17, 23). To investigate whether the chimeric i2/z has retained its ability to negatively regulate AC, we cotransfected the 293 cells with cDNAs encoding the rat LHR, -opioid receptor, and one of three  subunits (i2, z, or i2/z). Coexpression of LHR allows the targeting of transfected cells, and activation of this receptor by 5 ng/ml hCG raises the cAMP content to a level at which inhibition can be easily detected (23). In this system, 100 nM DPDPE was shown to inhibit consistently the hCG-stimulated cAMP production by 40-60% (20). In the current study, coexpression of z, but not i2, conferred PTX resistance to the DPDPE-induced inhibitory response (Fig. 2). In cells coexpressing the i2/z chimera, the DPDPE-mediated inhibition of cAMP accumulation was only slightly reduced by PTX treatment, suggesting that i2/z can indeed inhibit AC in an z-like manner (Fig. 2).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   PTX-insensitive inhibition of AC by the i2/z chimera. The 293 cells were cotransfected with the LHR cDNA (0.15 µg/ml), the -opioid receptor cDNA (0.25 µg/ml), and 0.15 µg/ml concentration of an  subunit (i2, z, or i2/z). Transfected cells were labeled with [3H]adenine in the absence or presence of PTX (100 ng/ml) and then assayed for cAMP accumulation in response to hCG (5 ng/ml) with or without DPDPE (100 nM). Data shown represent the mean ± standard deviation of triplicate determinations of one of three experiments with similar results. Results are expressed as percent inhibition of the hCG-stimulated activity in the presence of DPDPE compared with that measured in the presence of hCG alone. hCG-stimulated cAMP accumulation ranged from 13.3 ± 0.7 to 16.1 ± 1.6. *, DPDPE significantly inhibited the hCG response (paired t test, p < 0.05).

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Constitutive inhibition of AC by i2/z-QL and z/i2-QL. The 293 cells were cotransfected with the LHR cDNA (0.15 µg/ml) with or without 0.15 µg/ml pcDNA1 or one of the following  subunits in their wild-type (WT) or constitutively activated (QL) form: i2, z, i2/z, or z/i2. Transfected cells were assayed for cAMP accumulation in response to hCG (5 ng/ml). Data shown represent the mean ± standard deviation of triplicate determinations of one of three experiments with similar results. *, hCG responses were significantly inhibited in cells coexpressing the QL mutants (paired t test, p < 0.05).

Receptor coupling to t1/z and z/t1. Like i and z chains, t1 and t2 (rod and cone transducins, respectively) belong to the subfamily of G_i proteins. Although they are related, the transducins seem to couple exclusively to the opsin receptors and not to any of the G_i-coupled receptors. By constructing chimeras between t1 and z, we investigated whether the receptor recognition determinants of z are indeed located on its two termini. As shown in Fig. 4, coexpression of t1 attenuated the DPDPE-induced cAMP formation in the AC II transient transfection system. This is in agreement with the notion that t1 is a scavenger (25). Interestingly, blockade of the -mediated stimulation of AC II was also observed with cells coexpressing the t1/z chimera (Fig. 4). Both t1 and t1/z significantly inhibited the ability of DPDPE to stimulate AC II (p < 0.05, three experiments). By behaving like t1, the t1/z chimera seemed to be unable to release subunits when the -opioid receptor was activated. Lack of coupling to the -opioid receptor by t1/z was further demonstrated by its inability to provide PTX resistance to cells coexpressing this chimera (Fig. 4). On the contrary, the z/t1 chimera enhanced the DPDPE-induced stimulation of AC II. The potentiating effect of z/t1 suggested that this chimera can interact with the -opioid receptor and release subunits in addition to those released from endogenous Gi proteins. Because z/t1 contains the PTX-catalyzed ADP-ribosylation site, the DPDPE-induced AC II response was sensitive to PTX treatment (Fig. 4). Introduction of the QL mutation into z/t1 produced a constitutively active chimera that can inhibit AC in a receptor-independent fashion.² Taken together, these results indicate that the carboxyl-end 36 amino acids of t1 can substitute for cognate sequence on z but that sequence integrity at the amino terminal of z is also needed for recognition of G_i-coupled receptors.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Blockade of DPDPE-induced stimulation of AC II by t1 and t1/z chimeras. 293 cells were cotransfected with various cDNAs as described in the legend to Fig. 1, except that the following  subunits were used: t1, z, t1/z, or z/t1. Transfected cells were assayed for cAMP accumulation as for Fig. 1. Data shown represent triplicate determinations in a single experiment; two independent experiments yielded similar results. Results are expressed as percent stimulation of cAMP formation in the presence of DPDPE compared with that measured in the absence of DPDPE. The basal values are expressed as the ratio (×103) of cAMP to total adenine nucleotides and ranged from 5.71 ± 0.31 to 8.47 ± 0.44. *, DPDPE-stimulated cAMP accumulation was significantly different from that observed in the vector control (paired Bonferroni t test, p < 0.05). **, Significantly higher than basal cAMP accumulation in PTX-treated cells (paired t test, p < 0.05). Left, different wild-type and chimeric  subunits.

The -opioid receptor does not couple to 12/z or 13/z. It has previously been shown that G_i-coupled receptors can activate chimeric  subunits if the last few residues of the chimeras compose a "G_i-like" sequence (4, 29). These reports tend to place more weight on the importance of the carboxyl terminus of the  chains in receptor recognition and contradict our findings with the t1/z chimera. We therefore examined the ability of the -opioid receptor to stimulate AC II via subunits released from chimeric 12/z and 13/z subunits. Both chimeras have the last five amino acids changed to an z sequence (29). Cells were cotransfected with cDNAs encoding AC II, _s-Q227L, the -opioid receptor, and one of five  subunits (z, 12, 13, 12/z, and 13/z). The transfected cells were then assayed for cAMP accumulation in the absence or presence of 100 nM DPDPE. Except for cells coexpressing z, no enhancement of DPDPE-induced stimulation of AC II activity was seen with any of the transfected cells (Fig. 5). Moreover, only coexpression of z provided PTX resistance to the transfected cells. Hence, the -opioid receptor was unable to couple to 12 or 13, and the presence of the last five amino acids of z was insufficient to forge an interaction. Lack of coupling to 12 and 13 have already been demonstrated with the µ-opioid receptor (26).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Lack of -opioid receptor coupling to 12/z and 13/z chimeras. The 293 cells were cotransfected with various cDNAs as described in the legend to Fig. 1, except that the following  subunits were used: z, 12, 13, 12/z, or 13/z. Transfected cells were assayed for cAMP accumulation as for Fig. 1. Data shown represent triplicate determinations in a single experiment; two independent experiments yielded similar results. Results are expressed as percent stimulation of cAMP formation in the presence of DPDPE compared with that measured in the absence of DPDPE. The basal values are expressed as the ratio (×103) of cAMP to total adenine nucleotides and ranged from 5.83 ± 0.72 to 8.33 ± 0.68. *, DPDPE-stimulated cAMP accumulation is significantly different from that observed in the vector control (paired Bonferroni t test, p < 0.05). **, Significantly higher than basal cAMP accumulation in PTX-treated cells (paired t test, p < 0.05). Left, wild-type and chimeric  subunits used in the study.

Chimeric i2/z does not discriminate between various G_i-coupled receptors. Many receptors can apparently discriminate between different G proteins. For example, somatostatin receptor subtype 3 selectively couples to G_i1 rather than G_i2 or G_i3 (30), whereas the complement C5a receptor prefers G₁₆ to G₁₁ (31). There is no hard and fast rule that if a particular G protein interacts with one receptor, it will couple to other receptors of the same class. Hence, we examined the ability of i2/z chimera to interact with a panel of G_i-coupled receptors by using the same transient transfection strategy. Coexpression of either z or i2/z with the other two opioid receptor subtypes (µ and ) in 293 cells conferred PTX resistance in the stimulation of AC II activity by the opioid ligands tested (Table 1). In addition, both z and i2/z mediated PTX-insensitive stimulation of AC II on activation of the FMLP receptor (Table 1) (27). G_z is known to interact with receptors for aminergic neurotransmitters (17). Indeed, under similar experimental conditions, both ₂-adrenergic and dopamine D₂ receptors were able to activate the i2/z chimera leading to -mediated stimulations of AC II (Table 1). Collectively, these results suggest that the i2/z does not discriminate among different G_i-coupled receptors.

View this table: [in this window] [in a new window]   TABLE 1 Receptor-mediated stimulation of AC II via PTX-insensitive wild-type and chimeric  subunits The 293 cells were cotransfected with cDNAs encoding AC II (0.25 µg/ml), s-Q227L (0.025 µg/ml), 0.25 µg/ml concentration of a Gi-coupled receptor, and one of several  subunits: i2, i2/z, z, 12, 13, 12/z, or 13/z. The following receptors were tested: 2-adrenergic, dopamine D2, FMLP, -opioid, µ-opioid, and -opioid. Transfected cells were treated with PTX and assayed for cAMP accumulation in the absence or presence of receptor-selective agonists. Agonists used include 10 nM UK-14304 for 2-adrenoceptor, 10 µM quinpirole for dopamine D2 receptor, 200 nM FMLP for FMLP-receptor, 100 nM DPDPE for -opioid receptor, 100 nM [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin for µ-opioid receptor, and 100 nM U50,488 for -opioid receptor. Data shown represent mean ± standard error of three or four independent experiments of triplicate determinations. Results are expressed as percent stimulation of cAMP formation in the presence of agonist compared with that measured in the absence of agonist (basal). The basal values expressed as the ratio (×103) of cAMP to total adenine nucleotides and ranged from 4.77 ± 0.81 to 9.46 ± 0.85.

The 12/z and 13/z chimeras distinguish aminergic from peptidergic receptors. Although i2/z seemed to interact with a large variety of G_i-coupled receptors, the inability of the -opioid receptor to activate 12/z and 13/z suggested that the latter chimeras may not be as promiscuous. Activation of the Na⁺/H⁺ exchanger by the dopamine D₂ receptor via 13/z has been previously demonstrated (29). Coupling of the dopamine D₂ receptor to 13/z was therefore examined in the AC II transient transfection assays. The 293 cells cotransfected with AC II, s-Q227L, dopamine D₂ receptor, and 13/z were treated with PTX before cAMP assays. Activation of the dopamine D₂ receptor by 10 µM quinpirole increased cAMP accumulation by ~100% (Table 1). As a control for complete inactivation of endogenous G_i proteins by PTX, similar transfections were performed in which 13/z was replaced by i2. No agonist-induced stimulation of AC II was observed in control cells (Table 1), suggesting that the quinpirole response was indeed transduced by 13/z. Replacement of the dopamine D₂ receptor with the ₂-adrenoceptor in the cotransfections produced cells in which 10 nM UK-14304, an ₂-adrenoceptor agonist, stimulated AC II in a PTX-resistant manner (Table 1). However, when we tested the µ-opioid, -opioid, and FMLP receptors under the same experimental conditions, none of these peptidergic receptors were able to interact with 13/z (Table 1). The receptor-coupling profile of 12/z was essentially identical to that of 13/z (Table 1). Both 12/z and 13/z seemed to recognize aminergic but not peptidergic G_i-coupled receptor.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Immunodetection of chimeric  subunits. The 293 cells were transiently cotransfected with cDNAs encoding the AC II, s-Q227L, and -opioid receptor in the absence (Mock) or presence of various wild-type and chimeric constructs. Plasma membranes were prepared 48 hr after transfection. Membrane proteins (75 µg) were separated on a 12.5% polyacrylamide-sodium dodecyl sulfate gel and electrophoretically transferred to polyvinylidene difluoride membranes. Protein markers were localized by Ponceau S staining, and the chimeras were immunodetected with the specific antisera as indicated. Two independent experiments with different batches of membrane proteins yielded similar results.

	Discussion

Summary Introduction Procedures Results Discussion References

To achieve fidelity in signal integration and amplification, input from a large number of distinct receptors must be processed with precision by the G proteins. Specificity in G protein/receptor interactions has been postulated to reside at both the amino and the carboxyl termini of the G protein  subunit (1). Participation of the two termini as integral components of a putative receptor contact domain has been confirmed by studying the crystal structures of heterotrimeric G_t1 (12) and G_i1 proteins (11). Because numerous G_i-coupled receptors possess the ability to use G_z to mediate PTX-insensitive inhibition of AC, the receptor recognition domains of G_i and G_z must be highly similar. By comparison, the first 36 residues of i2 and z share ~63% identity, whereas the last 36 amino acids exhibit ~83% identity. The higher homology found between the carboxyl termini of i2 and z suggests that the carboxyl terminal region may be more critical for receptor recognition.

Our studies with the i2/z and z/i2 chimeras clearly demonstrate that the carboxyl termini of i2 and z are interchangable with respect to interaction with receptors. However, replacement of the carboxyl-terminal 36 residues of t1 with an z sequence (exhibiting ~75% identity) did not permit the resultant t1/z chimera to interact with G_i-coupled receptors. Hence, molecular determinants other than the carboxyl-terminal sequences are apparently required for the recognition of G_i-coupled receptors. Similar inferences can be drawn from studies using the z/t1 chimera. Because the z/t1 chimera was able to interact functionally with G_i-coupled receptors, the last 36 residues of t1 must be highly homologous to those of i2 and z. If the carboxyl terminus constitutes the only receptor contact site on t1, then G_t1 should be activatable by G_i-coupled receptors, yet G_t1 does not interact appreciably with G_i-coupled receptors (22, 26). Presumably, the amino-terminal regions of t1 may be more important for conferring specificity in the interaction of G_t1 with the rhodopsin receptor, whereas the carboxyl end of z (and i2) may play a more critical role in the interaction with G_i-coupled receptors.

A previous study using q chimeras illustrated the importance of the last five residues of i2 and z in the recognition of G_i-coupled receptors (4). If the first 30-40 residues of the  subunit contribute toward the formation of the receptor contact surface, then the amino termini of q, z, and i2 must bear substantial resemblance. However, sequence analysis of this region indicates ~50% identity between q and z (or i2) chains. A similar degree of identity can be found between the amino termini of t1 and z, but the t1/z chimera was unable to interact productively with G_i-coupled receptors. It seems that even when the carboxyl terminus contains the necessary residues, discrete sequence motifs within the amino-terminal region may dictate whether an  subunit can interact with G_i-coupled receptors. Because the extreme amino terminus of many  subunits contains sites for fatty acylation and covalent modifications and the putative receptor contact domain has substantial overlaps with the -binding surface (11, 12), it is difficult to determine which residues are absolutely critical for receptor recognition. Furthermore, G protein  subunit/receptor interactions may involve a series of conformational changes on association with the complex, which may add to receptor/G protein specificity and stabilization of interactions. At least for the two splice variants of G_o, the exact composition of the subunits decrees which G_i-coupled receptors can be recognized by G_o (32, 33). Moreover, failure to properly bind subunits may result in the inability of the  subunit to interact with receptors (34).

The structural integrity of chimeras composed of i2 and z sequences deserves further comment. We have shown not only that the i2/z and z/i2 chimeras can interact with a variety of G_i-coupled receptors nut also that they can adopt an active GTP-bound conformation to inhibit AC. Additional support can be drawn from the fact that PTX abolished the interaction between receptors and z/i2, suggesting that the z/i2 chimera can maintain a structure recognizable by the toxin. The PTX sensitivity of z/i2 is in direct contrast with an s/i2 chimera that does not serve as a substrate for PTX (35). Because the amino and carboxyl termini of the  subunit lie in close proximity, replacement of the amino-terminal sequence of i2 with an s sequence may disrupt the PTX recognition surface, whereas the z sequence is sufficiently homologous to i2 that PTX can ADP-ribosylate the z/i2 chimera. By the same analogy, structural integrity of the z/t1 chain must be properly preserved because this chimera can interact with receptors in a PTX-sensitive manner.

Analysis of the 12/z and 13/z chimeras revealed an interesting profile of receptor coupling specificity. Aminergic receptors were able to interact with both 12/z and 13/z chimeras, albeit at a weaker efficacy than with the i2/z chimera. The peptidergic receptors, on the other hand, were totally unable to interact with the 12/z and 13/z chimeras. It remains to be determined whether the difference in coupling to 12/z and 13/z chimeras is generally applicable to all G_i-coupled aminergic and peptidergic receptors. The lack of coupling to peptidergic receptors was not due to inadequate expression of 12/z and 13/z. In addition, each of the peptidergic receptor tested was apparently expressed in sufficient quantities to interact with both z and i2/z. Although we cannot exclude the possibility that coexpression with 12/z or 13/z may affect the expression of peptidergic receptors, this explanation is probably incorrect because the expression of FMLP receptors (as determined by radioligand binding assays) was unaffected by the type of  subunits being coexpressed (27). It is more likely that the 12/z and 13/z chimeras are incapable of interacting with peptidergic receptors. The precise molecular determinants that specify receptor/G protein interactions are not well understood. For a receptor to couple efficiently to a G protein, the respective contact surfaces on both components must be complementary to each other. A G_q family member, G₁₆, has recently been contrived as an universal adapter for G protein-coupled receptors (36). The  subunit of G₁₆ may have the most "fitting" or "flexible" receptor contact surfaces among the G proteins. By constructing chimeras composed of 16 and 11 (which does not interact with G_i-coupled receptors) sequences, it has been shown that the specificity of G₁₆ coupling to the C5a receptor is primarily found within the amino-terminal 209 residues of 16 (31). Whether the same regions are also necessary for 16 to interact with other receptor types remains to be resolved. By building on the information obtained from chimera studies and crystal structures of heterotrimeric G proteins, further investigations may provide insights on the minimum molecular determinants for  subunits to interact with receptors.