Introduction

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The signaling properties of  subunit of G_z protein (G_z) are very similar to the three G_i subtypes. All G protein-coupled receptors that have been shown to interact with G_z are known couplers of G_i proteins (Fields and Casey, 1997; Ho and Wong, 1998). Although the overall amino acid identity of G_z and G_i2 is about 60%, they inhibit adenylyl cyclase (AC) in a similar fashion (Wong et al., 1992; Kozasa and Gilman, 1995). The sequence identities are even higher when only their C-terminal halves are compared, where the putative receptor- and effector-interacting domains were located. However, subtle differences are found between the amino acid sequences of G_i2 and G_z, which suggest that they may use different structural elements to achieve similar functions. Incorporation of the last five residues of either G_i2 or G_z, which shared very low homology to each other, to the corresponding region of G_q broadened its receptor coupling profile to both G_q- and G_i-linked receptors (Conklin et al., 1993). However, the replacement of the last 36 amino acids of G_z with those of G_t1 did not block the coupling of the resultant G_z/G_t1 to G_i-linked receptors (Tsu et al., 1997). Indeed, a number of studies suggested that the amino terminus of G subunit might be crucial for receptor coupling (Hamm et al., 1988; Kostenis et al., 1997). Furthermore, a stretch of amino acids, 220 to 240 of G₁₆, has been shown to be essential for receptor coupling (Lee et al., 1995). It prompted us to identify the essential determinants of G_z for specifying its receptor coupling property.

The effector interacting domains of various G subunits are generally localized at the C-terminal half of the amino acid sequence (Berlot and Bourne, 1992; Medina et al., 1996; Grishina and Berlot, 1997). Although G_s and G_i2 regulate AC in opposite fashions, they have evolved similar and different stretches of amino acids for effector interactions (Berlot and Bourne, 1992; Grishina and Berlot, 1997). The AC inhibiting residues of G_i2 were localized at the Switch II region (similar to G_s) and 4/6 loop (unlike G_s) by alanine mutagenesis (Grishina and Berlot, 1997). It is unclear if regions similar to those found in G_i2 are responsible for effector interaction in G_z. Interestingly, mutations of the acylation-modified N-terminal residues altered the constitutive inhibitory action of G_z to AC (Wilson and Bourne, 1995). Moreover, alanine substitution of the protein kinase C-phosphorylation sites of mutationally active G_z attenuated its inhibitory effect on AC (Ho and Wong, 1997). It is still possible that the N-terminus of G_z is also involved in effector regulation.

We attempted to localize the receptor interacting as well as AC inhibiting domains of G_z by constructing chimeric G subunits using G_z and G_t1. G_t1 is approximately 60% identical with G_z; hence, their tertiary structures should have considerable resemblance. Within the G_i-subfamily, G_t1 is primarily coupled to rhodopsin and regulates cGMP phosphodiesterase. Introduction of G_t1 portions into a G_z backbone would be expected to have minimal interference on the protein folding and AC inhibition. Desired recombinant proteins were transiently expressed in human embryonic kidney (HEK) 293 cells and the functions of the chimeras were assessed in two aspects. To study the receptor coupling events, the abilities of chimeric G subunits to couple to -opioid receptor (DOR) were monitored by the -mediated stimulation of type 2 adenylyl cyclase (AC2). For studying the effector interaction, the abilities of the chimeras to inhibit isoproterenol-stimulated cAMP accumulation upon the activation of DOR were monitored. Another series of chimeras were constructed with a point mutation that rendered GTPase-deficient phenotypes (Freissmuth and Gilman, 1989; Graziano and Gilman, 1989). They were applied to investigate the constitutively inhibitory effects of the chimeras on isoproterenol-stimulated AC activity.

	Experimental Procedures

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Materials. AC2 and DOR cDNA were gifts from Randall Reed (Johns Hopkins School of Medicine, Baltimore, MD) and Christopher Evans (University of California, Los Angeles, CA), respectively. Pertussis toxin (PTX) was purchased from List Biological Laboratories, Inc. (Campbell, CA). HEK 293 cells were obtained from the American Type Culture Collection (Rockville, MD). [d-Pen^2,5]enkephalin (DPDPE) was from Research Biochemicals Inc. (Natick, MA). Antisera against G_z and G_t1 were from Gramsch Laboratories (Schwabhausen, Germany) and Transduction Laboratories (Lexington, KY), respectively. [³H]Adenine was purchased from Amersham (Buckinghamshire, UK). Plasmid purification columns were obtained from Qiagen (Hilden, Germany). Taq DNA polymerase, customized primers, restriction endonucleases, and cell culture reagents were obtained from Life Technologies Inc. (Grand Island, NY). All other chemicals were purchased from Sigma (St. Louis, MO).

Construction of Chimeric  Subunits. A complete list of chimeric and mutational G subunits is shown in Table 1. The chimeras zt40 and zt43 were constructed by polymerase chain reaction (PCR) using a pair of chimeric primers (Table 2). The full-length PCR product was sequenced using Sequenase Version 2.0 DNA sequencing kit from Amersham. Other chimeras were constructed by making use of convenient unique restriction sites and exchanging the corresponding restriction fragments of G_t1, zt40, or zt43 with G_z (Table 1). For example, the junctional restriction sites of tz36, tz60, and tz143 are BglII, AflIII, and BamHI, respectively. The insertion mutant +4t and PTX-resistant G_t1CG mutant were made by PCR using G_t1 as the template, and +4t/CG is generated by combining +4t and G_t1CG together through restriction cutting. 4z Is derived from G_z with the residues 11 to 14 deleted. For si143 and sz143, the portion of G_s was obtained by partial digestion with BamHI and then ligation with the C-terminal portion of either G_i2 or G_z. GTPase-deficient mutants of various chimeras were made by replacing G_t1 and G_z with G_t1RC (Ho et al., 1999) and G_zQL (Wong et al. 1992), respectively in the construction procedures. All the cDNA constructs were subcloned in the mammalian expression vector pcDNAI at EcoRI site.

View this table: [in this window] [in a new window]   TABLE 1 List of mutational and chimeric G subunits Except for the last three constructs, the nomenclatures of the chimeras are according to the parental G subunits (templates) and the numbers of amino acids of Gz present in the chimeras. All chimeras are constructed either by PCR using the primers listed in Table 2 or by restriction digestion (RD) of templates and subsequent ligation. The restriction sites shown are the junctional sites of the ligated fragments.

Transfection of HEK 293 Cells and cAMP Accumulation Assay. HEK 293 cells were cultured with Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum (v/v), 50 U/ml penicillin, and 50 µg/ml streptomycin at 37°C in humidified air with 5% CO₂. They were cotransfected with various recombinant DNA constructs using DEAE-dextran/chloroquine method as described previously (Wong, 1994). Transfected cells were labeled with 1 µCi/ml of [³H]adenine in MEM with 1% fetal calf serum and treated with 100 ng/ml PTX as appropriate. Labeled cells were treated with proper receptor agonists in 20 mM HEPES-buffered MEM with 1 mM 1-methyl-3-isobutylxanthine for 30 min and the reactions were terminated by adding ice-cold 5% trichloroacetic acid with 1 mM ATP. Separation of labeled cAMP from other nucleotides was achieved by sequential ion exchange chromatography as described previously (Salomon, 1991). The cAMP levels were interpreted as the ratios of the counts per minute of [³H]cAMP fractions to those of [³H]ATP fractions and expressed as [cAMP/(cAMP + Total) × 1000]. Absolute values for cAMP accumulation varied between experiments, but variability within a given set of transfection was in general <10%. Data shown in the figures were the mean ± S.E.M. of three to five individual experiments performed in triplicate. ANOVA and paired t test with 95% confidence was used to analyze the significance between different treatment groups.

Western Blotting Analysis. Crude membrane proteins from HEK 293 cells transfected with various chimeric G subunits were extracted as described previously (Ho and Wong, 1997). Each protein sample (50 µg) was resolved in 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membrane. Antiserum 3A-170 (Gramsch Laboratories, Schwabhausen, Germany) against the carboxyl terminus of G_z was used for the detection of G_z, tz, and ztz chimeras, whereas G_t1, zt40, and zt43 were identified with anti-G_t1 antiserum (Transduction Laboratories).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

The Role of the C-Terminus of G_z in Coupling with DOR. The abilities of chimeric G subunits to couple to DOR were monitored by the -mediated stimulation of AC2. This reporter system is advantageous because it could be generally applied to assess the coupling between different categories of receptor-G protein pairs without considering the functions of the G subunits. Moreover, AC2 is relatively insensitive to G_i-mediated inhibition (Taussig et al., 1994). It eliminates the possibility that G_z and complex act on AC2 in an antagonistic fashion. The AC2 system is especially useful for checking the receptor coupling efficiencies of particular chimeric G subunits that have lost the integrity of effector interacting domains. A schematic representation of the first series of chimeric G subunits was shown in Fig. 1A. We attempted to examine the importance of the C-terminal tail of G_z on receptor coupling using this series of chimeras. In the vector control, HEK 293 cells expressing G_sQL, DOR, and AC2 were treated with or without PTX. Addition of 100 nM DPDPE stimulated the cAMP production to about two times the basal level in the absence of PTX treatment (Fig. 1B). The DPDPE-induced enhancement was abolished in cells treated with PTX, indicating that the coupling between DOR and the endogenous G_i proteins was obstructed. Coexpression of recombinant G_z provided a larger pool of G protein trimers capable of coupling to DOR; hence, in the absence of PTX treatment, a 150% enhancement of the cAMP production was observed compared with the corresponding response in vector control. PTX treatment partially reduced the cAMP level of G_z-expressing cells, which suggested that G_z could functionally replace the role of endogenous G_i and mediated the DPDPE-induced cAMP production in a PTX-resistant manner. In the case of G_t1, the response in the absence of PTX treatment was significantly lower than that of vector control and was close to the basal value. Suppression of -mediated AC2 activity was caused by the strong -scavenging property of G_t1. Moreover, there was no increase of cAMP level in cells treated with PTX, which inactivated endogenous G_i and the recombinant G_t1. These results are consistent with those reported earlier (Tsu et al., 1997).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   The first series of chimeric G subunits and receptor-mediated regulation of AC2. The parental (Gz and Gt1) and chimeric G subunits are diagrammatically shown in A. B, mutationally active GsQL (0.025 µg/ml), AC2 (0.25 µg/ml), DOR (0.25 µg/ml), and 0.25 µg/ml of one of the G subunits in A were coexpressed in HEK 293 cells. Transfected cells were treated with or without 100 ng/ml PTX as indicated. cAMP levels were measured in the absence or presence of 100 nM DPDPE. Asterisks indicate the DPDPE-induced cAMP levels are significantly higher than those of vector control.

The N-Terminus of _z Is Essential for Receptor Coupling. Recent studies highlighted the importance of the N-terminus of G subunit for receptor specificity (Kostenis et al., 1997). A second series of chimeras were thus constructed to verify the role of the N-terminus of G_z on DOR coupling. The chimera zt40 was constructed so that the N-terminal helix became G_z-like (Fig. 2A, the third construct). In the absence of PTX, DPDPE-induced enhancement of cAMP accumulation in zt40-transfected cells resembled that of G_z-transfected cells. However, zt40 is PTX-sensitive because it acquired the C-terminus of G_t1 and the DPDPE-induced cAMP production was abolished in the presence of PTX (Fig. 2B). This result suggested that the N-terminal region of G_z was an essential determinant for receptor coupling. The chimera ztz40/36 was then constructed based on zt40 and tz36 (Fig. 2A, the fourth construct) to check whether the inclusion of the G_z-specific C-terminus would confer PTX resistance to the ztz40/36 chimera. The phenotype of ztz40/36 was more or less the same as G_z, except that the percentage response over basal value was slightly lower than that of G_z (Fig. 2B). It suggested that the C-terminus of G_z only conferred PTX-resistance but did not further enhance the receptor coupling efficiency. Two other ztz chimeras, ztz40/60 and ztz40/143 (Fig. 2A, sixth and seventh constructs), were also able to couple to DOR and stimulate AC2 to slightly greater degree than ztz40/36 (Fig. 2B).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   The second series of chimeric G subunits and receptor-mediated regulation of ACs. The parental (Gz and Gt1) and chimeric G subunits are diagrammatically shown in A. B, HEK 293 cells were transfected, labeled, and treated as indicated in the legend to Fig. 1B. Basal cAMP levels ranged from 4.11 ± 0.33 to 6.72 ± 0.18. Single and double asterisks indicate that the basal and DPDPE-induced cAMP levels, respectively, are significantly higher than those of vector control. C, HEK 293 cells were cotransfected with 2AR (0.15 µg/ml), DOR (0.025 µg/ml), and 0.25 µg/ml of one of the G subunits in A. Transfected cells were all treated with 100 ng/ml PTX. cAMP production was triggered by treating the cells with 10 µM isoproterenol alone or in the presence of 100 nM DPDPE. DPDPE-induced inhibition was expressed as percentage inhibition of the isoproterenol-stimulated cAMP levels that ranged from 10.55 ± 1.78 to 14.08 ± 1.24. Asterisks indicate the DPDPE-induced inhibition of cAMP production is significantly greater than that of vector control.

The Amino Acids 11 to 14 of _z Are Essential for Receptor Coupling. The N-terminus of G_t1 is considerably divergent from those of G_i2 and G_z in two aspects. First, the N-terminus of G_z is more homologous to G_i2 (47.1%) than to G_t1 (33.3%). Second, the length of the theoretical N-terminal helix of G_t1 is shorter than G_i2 or G_z by four amino acids (Fig. 3A). Progressive deletion of the six N-terminal residues of G_q broadened its receptor coupling specificity (Kostenis et al., 1997). It is conceivable that the length of the N-terminal region of the chimeras may affect receptor activation. In the case of zt40, its ability to couple to DOR may be attributable to the elongation of its N-terminal region. To test this possibility, an insertion mutant of G_t1 was constructed by inserting the four residues of G_z between residues 10 and 11 of G_t1. The cysteine residue at 4 position of the C-terminus of G_t1 was mutated into glycine and the G_t1 mutant (termed +4t/CG) became resistant to PTX-mediated inactivation. This mutation allowed us to examine the coupling of +4t/CG with DOR without the interference caused by endogenous G_i proteins. In the presence of PTX, application of DPDPE can induce a modest increase of cAMP level in +4t/CG-transfected cells, which is significantly higher than that of G_t1CG- or vector-transfected cells (Fig. 3B). It indicated that +4t/CG can indeed couple to DOR in a PTX-insensitive manner although the response is relatively weak (~40% increase as compared with the basal). To further confirm the role of these four amino acids, a deletion mutant of G_z was constructed with the amino acids 11 to 14 removed (termed 4z) and the mutant was tested for its ability to recognize DOR. In AC2 assays, 4z-transfected cells showed only slight reduction of DPDPE-induced cAMP accumulation compared with G_z (Fig. 3B). The reduction of response was also observed when the cells were pretreated with PTX. It suggested that residues 11 to 14 of G_z form one of the minor determinants for DOR coupling. The reduction of DOR-induced inhibitory response in 4z-transfected cells is probably independent to its expression level (Fig. 4) and effector interaction. A constitutively active mutant of 4z, 4z/QL, inhibited isoproterenol-induced cAMP production in the cells cotransfected with ₂-adrenoceptor to a similar extent as G_zQL (Fig. 3C). Overall, these results strongly suggested that the N-terminal residues 11 to 14 of G_z are involved in receptor recognition.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Amino acids 11 to 14 of Gz are essential for DOR coupling. A, the sequences of the two termini of Gz, Gt1, and three mutants were aligned. Deletion of the residues 11 to 14 of Gz yielded the mutant 4z. A cysteine-to-glycine mutation was introduced to the 4 position of Gt1 to form Gt1CG. +4t/CG was made by inserting the residues 11 to 14 of Gz between residues 10 and 11 of Gt1CG. Underlined residues are the mutated and inserted residues. B, HEK 293 cells were transfected, labeled, and treated as indicated in the legend to Fig. 1B. Basal cAMP levels ranged from 4.51 ± 0.73 to 5.45 ± 0.44. Single and double asterisks indicate that the basal and DPDPE-induced cAMP levels, respectively, are significantly higher than those of vector control. C, HEK 293 cells were cotransfected with 0.15 µg/ml of 2AR and 0.25 µg/ml of one of the G subunits. cAMP production was triggered by treating the cells with 10 µM isoproterenol. The cAMP levels were expressed as percentage responses of Gz. WT, wild type; QL, active mutant.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Western blotting analysis of chimeric G subunits. 0.25 µg/ml of each chimeric G subunit cDNA was transfected into HEK 293 cells and membrane proteins were extracted for immunodetection. Antisera specific against Gt1 and Gz were applied for blots in A and B, respectively. Two individual experiments using different batches of protein samples showed similar results.

Protein Expression of Chimeric G Subunits. Membrane proteins of the cells transfected with various chimeras studied here were separated by denaturing gel electrophoresis and the chimeras were detected by specific antisera. The expression levels of zt40 and zt43 were similar to tz36, but lower than G_t1 in HEK 293 cells as detected by an anti-G_t1 antiserum (Fig. 4A). Both G_t1CG and +4t/CG were expressed strongly in HEK 293 cells. Detection with an antiserum against the last 15 residues of G_z facilitated direct comparison of the expression levels of various chimeras (Fig. 4B). All chimeric  subunits were expressed to similar levels in HEK 293 cells as G_z. The results indicated that most of the chimeras studied here are expressed to similar levels.

Receptor-Mediated Inhibition of AC by Chimeric  Subunits. Some of the chimeras contained increasing lengths of C-terminal tails of G_z, and could serve as useful tools for localizing the AC-inhibiting regions on G_z. Thus, we tested these chimeras for their ability to inhibit AC. HEK 293 cells transfected with ₂-adrenoceptor, DOR, and a parental or chimeric G subunit were pretreated with PTX and then stimulated with 10 µM isoproterenol to elevate the intracellular cAMP level. Coadministration of DPDPE to the cells expressing G_z showed about 30 to 40% reduction of isoproterenol-induced cAMP level, which was not observed in the cases of vector control or G_t1-transfected cells (Fig. 2C). Both zt40 and zt43-transfected cells lacked DPDPE-induced inhibition of AC activity. The N-terminal G_z-specific sequence of these two chimeras did not contain sufficient structural elements for AC inhibition. Next, we examined the six ztz chimeras, because it has been shown that the N-terminal region of G_z was important for the coupling to DOR. ztz40/36 and ztz43/36 showed no significant inhibitory effect on the cAMP levels (Fig. 2C). Chimeras containing 60 or more C-terminal amino acids of G_z inhibited AC significantly. Such results indicated that one of the essential AC inhibitory domains must be located within amino acids 296 to 319 of G_z.

Inhibition of AC by Constitutively Active Chimeric G Subunits. Introduction of a point mutation at the GTP-binding regions of G subunit created receptor-independent constitutively active mutants (Freissmuth and Gilman, 1989; Graziano and Gilman, 1989). This approach has been successfully applied for studying the effector interacting domains of various G subunits even when the receptor interacting domains were disrupted in the chimeras (Medina et al., 1996; Grishina and Berlot, 1997). For the constructs used in this study, Arg-174 of the G_t1 domain and Gln-205 of the G_z portion were mutated to cysteine and leucine, respectively, and these chimeras were used to examine the receptor-independent constitutive inhibition of AC activity. Results are summarized in Fig. 5. As a positive control, the G_z mutant G_zQL (Wong et al., 1992) inhibited isoproterenol-stimulated cAMP level by 40 to 50% compared with the vector control, whereas G_t1 exhibited no observable inhibition on the AC activity. Among the three tz chimeras, tz60(RC) and tz143(RC) inhibited AC significantly, albeit to a lesser extent than that of G_zQL. Among the three zt chimeras, only zt319(QL) inhibited AC efficiently. Of the six ztz chimeras, only fourztz40/60(RC), ztz40/143(RC), ztz43/60(RC), and ztz43/143(RC)were able to inhibit AC significantly. The degrees of inhibition for all these chimeras were comparable with each other but slightly weaker than G_zQL. The protein expression levels of all these constitutively active chimeras were similar to each other and also comparable with those of their wild-type counterparts (Fig. 4). Collectively, the results confirmed the essential role of amino acids 295 to 319 of G_z for AC inhibition because all chimeras that possess the ability to inhibit AC contained this stretch of residues from G_z.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Constitutive inhibition of AC by mutationally active chimeric G subunits. HEK 293 cells were cotransfected with 0.15 µg/ml of 2AR and 0.25 µg/ml of one of the G subunits. cAMP production was triggered by treating the cells with 10 µM isoproterenol. The cAMP levels of the chimera-transfected cells were expressed as percentage inhibition of the mean value of the isoproterenol-stimulated cAMP levels of the vector control. Asterisks indicate the cAMP levels are significantly greater than the vector control.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

G_z is not simply a PTX-resistant substitute of the three G_i subtypes but it may be involved in a number of novel signaling events. The specific functional interactions of G_z with newly discovered proteins such as RGSZ1 (Glick et al., 1998), Rap1GAP (Meng et al., 1999), and GRIN1 (Chen et al., 1999) suggested that G_z has its distinct roles in signal transduction in addition to the regulation of ACs. Localization of the functional domains of G_z could provide valuable information for a better understanding of the roles of G_z in different molecular events. The present study investigated the receptor and AC-interacting domains of G_z. Two major findings could be concluded. First, the N-terminal sequence of G_z seemed to be a critical determinant for the coupling to DOR. Second, one of the AC inhibiting domains of G_z is located at amino acids 296-319.

A series of chimera studies indicated that the last five amino acids of G subunits are essential for determining the receptor specificity (Conklin et al., 1993, 1996). The last five amino acids of G_t1 are identical with those of G_i2 (Fig. 6), yet G_t1 primarily couples to rhodopsin. Both an evolutionary trace analysis (Lichtarge et al., 1996) and an extensive mutagenesis study (Onrust et al., 1997) suggested that residues spanning the whole 5 helix of the GTPase domain of G subunit interacts with receptor. Moreover, a recent study indicated that the intramolecular interaction between the GTPase and helical domains (4/3 and G/4 loops) of G subunit is important for receptor-mediated activation (Marsh et al., 1998). The series of tz chimeras retaining up to 143 C-terminal G_z residues essentially covered all the regions identified in the previous studies for receptor-mediated activation. The lack of DOR coupling with tz chimeras clearly indicated that the N-terminal half of G subunit also contain essential elements for receptor coupling.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Alignment of the amino- and carboxyl-terminal regions of Gi2, Gt1, and Gz. Portions of the amino acid sequences of Gi2, Gt1, and Gz were extracted from their complete alignment using CLUSTAL X program (numbers in brackets indicate the positions of the residues; Jeanmougin et al., 1998). The strictly and homologously conserved amino acids are marked with asterisks and dots, respectively, at the bottom of the aligned sequences. A, inverted and underlined residues are essential and nonessential for receptor coupling, respectively (Conklin et al., 1993; Beck et al., 1997; Ho and Wong, 1997; Onrust et al., 1997). B, inverted and underlined residues are important and unrelated to effector interactions, respectively (Faurobert et al., 1993; Spickofsky et al., 1994; Grishina and Berlot, 1997).

Alignment of the amino acid sequences of G_i2, G_z, and G_t1 yielded useful information (Fig. 6). The most interesting point is that the N-terminal helix of G_t1 is 4 amino acids shorter than G_i2 and G_z, but the charge distribution of the aligned residues are reasonably similar. A fair prediction would be the length of the N-terminal helix determined the receptor coupling efficiency. The phenotypes of the mutants +4t/CG and 4z (Fig. 3) supported the importance of N-terminus of G subunit in receptor coupling. Recent studies on the receptor coupling of G_q also support this notion. Deletion of the N-terminal six amino acids of G_q allowed the mutant to be activated by G_i-linked receptors (Kostenis et al., 1997). Alignment of the N-terminal sequences of various G subunits showed that the first six residues were unique for G_q. Indeed, the sequence identities and lengths of the N-terminal helices of different G subunits are very divergent and may be related to the peculiarities of the functions of each G subunit. For example, the N-terminal residues of G_q are involved in membrane attachment (in addition to the palmitate attached) and phospholipase C activation (Hepler et al., 1996). In one of our previous studies (Ho and Wong, 1997), mutation of the two protein kinase C-phosphorylation sites at the N-terminus of G_z abolished its constitutive inhibitory effect on AC. Further studies on the roles of N-terminal residues of other G subunits should provide more insights on their specific functions.

The crystal structures of trimeric G proteins (Wall et al., 1995; Lambright et al., 1996) provided clear evidence that the N-terminus of G subunit is one of the major -interacting sites. An early study of the interaction between G_o and subunits suggested that the reduction of the length of the N-terminus of G_o by four residues (positions 7-10) diminishes its binding to subunits (Denker et al., 1992). Preservation of the integrity of the N-terminal helix seems to be necessary for binding subunits properly. In the present study, changes in the ability of the chimeras to associate with the subunits might affect their coupling efficiencies to DOR. Moreover, it has been suggested that the N-terminal helix of G subunit is sandwiched by G subunit and receptor (Wall et al., 1995; Lichtarge et al., 1996), and it may play a role in transmitting the conformational changes of ligand-bound receptor to the subsequent intra-and intermolecular conformational changes of G protein trimer. Additional studies are required to address these possibilities.

One of the well-characterized representatives in the G_i-subfamily in effector interaction is G_i2, which is probably the closest member to G_z. Berlot and coworkers have identified a region of 79 amino acids of G_i2, which was sufficient to convey the AC inhibition (Medina et al., 1996). The residues of G_i2 for effector regulation were resolved eventually using alanine mutagenesis by the same laboratory (Grishina and Berlot, 1997) and they are localized on two structural elements of G_i2, the Switch II region and the 4/6 loop. Our results coincided with the findings in G_i2, the amino acids 291 to 314 of G_z actually corresponded to 4/6 loop on the GTPase domain of G_z. The same loop structure (but not the same residues at the corresponding positions) in G_s, G_i2, and G_t1 has also been shown to be an effector interacting region (Berlot and Bourne, 1992; Spickofsky et al., 1994; Grishina and Berlot, 1997; Natochin et al., 1999). It is interesting that different categories of G protein-regulated effector enzymes have evolved to interact with the similar structural elements of various G subunits. The 4/6 loop may be more than just a structural element bearing a single function. Using G_t1/G_i1 chimeras, Bae et al. (1997) showed that the area bounded by the 4 helix and the 4/6 loop of G_i1 is important for the coupling of 5-HT_1B receptor. Subsequent studies by the same group (Bae et al., 1999) demonstrated that two 4 helical residues in G_i1 (Gln-304 and Glu-308) are critical determinants of receptor-G protein coupling. In the present study, the incorporation of the 4/6 loop of G_z in the G_t1 backbone (such as tz60 and tz143) did not allow coupling to DOR. Although the 4 helix of G_z might be involved in receptor recognition, it alone was insufficient to support coupling to DOR.

We did not investigate further the importance of the Switch II region of G_z on AC inhibition because G_t1 and G_z have sequences identical with G_i2 at that region (Fig. 6) and so the corresponding region of G_t1 could provide essentially the same effector interacting residues. Inhibition of AC by G_z was not related to the N-terminal sequence of G_z because the constitutively active chimera tz60(RC) already exerted the inhibitory action (Fig. 4). The contribution of the variation of the G-1 GTP-binding region of G_z to the regulation of AC was only minimal (Fig. 2C). It implied that the slow GTP hydrolysis rate of G_z might not be related to the abilities of the chimeras to inhibit AC.

In conclusion, this report provides clear evidence that the N-terminal helix of G_z (and possibly that of G_i) is crucial for receptor-mediated activation. The length of the N-terminal helix is also critical for determining the efficiency of receptor coupling. The AC-inhibiting domains of G_z seem to be very similar to those of G_i2, including the 4/6 loop and Switch II region. Our results strongly suggested that G_z inhibited AC in a fashion similar to other inhibitory G subunits.