Function of a Heterologous Muscarinic Receptor in T Cell Antigen Receptor Signal Transduction Mutants*

Previously we have described a system of somatic cell genetics (J.CaM1 and J.CaM2) for analyzing signal transduction via the T cell antigen receptor com- plex (CD3/Ti). Here we describe a third mutant, J.CaM3, which also expresses high levels of receptors that are functionally impaired. Like J.CaM1, J.CaM3 demonstrates partial signal transduction via CD3/Ti to only certain stimuli. J.CaM1, J.CaM2, and J.CaM3 define three non-Ti complementation groups involved in receptor function. To evaluate the mutations further we have introduced a heterologous receptor, the human muscarinic receptor 1 (HMl), into the parental Jurkat and mutant cell lines. This receptor demonstrates signal transduction competence in all these hosts, indicat- ing that 1) T cells express the necessary apparatus for the coupling of HM1 to second messenger generation and 2) the mutations in the J.CaM family all affect molecules that are specific to CD3/Ti, and not HM1, function. Finally, the HM1 receptor exhibits partial sensitivity to cholera toxin in Jurkat cells, in contrast to the virtually complete sensitivity of CD3/Ti to chol- era toxin. The human T cell antigen receptor is among the large class of receptors that utilize the inositolphospholipid second messenger system to initiate cellular activation (1). Either the recognition of foreign antigens in the appropriate major histocompatibility context or the binding of monoclonal antibod-ies microfar-ads). A 300-V electric field pulse was discharged across a 0.4-cm path length cell suspension (lo7 cells/ml in 20 mM Hepes, pH 7.05, 137 mM NaCl, 5 mM KC], 0.7 mM Na2HP04, 6 mM dextrose). Selection of transfectants was begun 2 days after transfection in medium containing geneticin (GIBCO), 2 mg/ml. Saturation Binding AnnLysis- Binding studies for quantitating muscarinic receptor expression were performed with intact cells (IO6/ sample) using the muscarinic receptor antagonist [3H]quinuclidinyl benzilate ( [3H]QNB, 42 Ci/mmol, Amersham Corp.). Nonspecific binding was determined in the presence of 10 p~ atropine (Sigma). Incubations were performed at room temperature in 50% RPMI 1640, 50% phosphate-buffered saline, 2 mM EGTA. [3H]QNB binding reached a plateau by 90 min, at which time bound activity was determined by filtering the reaction mixture through a glas fiber filter (Whatman GF/B) under vacuum. The filter was washed twice with 5 ml of the incubation buffer and once with 5 ml of ice-cold phosphate-buffered saline. Bound radioactivity was determined by liquid scintil- lation counting of the filter. All incubations were performed in triplicate, and the mean binding at each concentration was used in the curve fitting. Binding curves were analyzed by the Eadie-Hofstee L. Lanier, personal communication.

( 1 Presently in the National Institutes of Health Medical Scientist largely unknown, although both conformational and receptor aggregation mechanisms have been proposed in other receptor systems. The T cell antigen receptor is an elaborate multimolecular structure (2,3). Gene transfer studies have demonstrated that the heterodimeric Ti subunit (consisting of the disulfidelinked (Y and @ integral membrane glycoproteins) is responsible for recognition of antigen-major histocompatibility complex (4,5). This function is consistent with the highly variable primary structure of the distal extracellular domains of the Ti chains. Still unknown is the precise function of the four or more invariant integral membrane proteins (the CD3 complex) (6) that appear to have obligate, noncovalent association with the Ti subunit (7,8).
Because of the molecular complexity of the CD3/T' 1 complex, we have been utilizing a system of somatic cell genetics for studying structure/function relationships within the receptor-mediated signal transduction pathway. We have described two independently derived somatic cell mutants isolated from the Jurkat human leukemic T cell line (9, 10).
While both express high levels of grossly normal antigen receptor complexes on the cell surface, they exhibit only partial (J.CaM1) or entirely absent (J.CaM2) coupling between CD3/Ti and phosphoinositide (PI) second messenger production. We recently demonstrated that both cell lines have non-Ti mutations that lie in different complementation groups, indicating that at least two non-antigen-binding molecules are critical to signal transduction by the receptor (10). The partial integrity of signaling function in response to some anti-CD3 (but not anti-Ti) mAbs in J.CaM1 led us to propose that CD3 normally is coupled directly to the second messenger system and that signal transduction involves transfer of information from Ti to CD3 and from CD3 to the next component in the pathway.
Of great interest in elaborating the molecular events underlying signal transduction by CD3/Ti is the identification of the mutant molecules in J.CaM1 and J.CaM2. Since a priori there are too many candidates to permit searching at the genetic level, one rational approach is to begin to exploring the functional consequences of the mutations on other receptor systems. For example, we found previously that a second T lineage-specific surface receptor (CD2) that is coupled to PI metabolism is also impaired in the J.CaM1 line, which had been selected specifically for defects in CD3/Ti function (11). These and other studies (12) suggested that CD2 function converges with that of CD3/Ti proximally in the signaling pathways. Since CD2 also depends functionally on surface expression of CD3/Ti, however, the relationship between these molecules appears to be complex. For further investigation of the pathways leading to second messenger production, we have now utilized a heterologous PI-coupled receptor (the human muscarinic subtype 1 receptor, HM1) for gene transfer studies of the phenotypes of J.CaM1, J.CaM2, and a recently derived mutant, J.CaM3.
For analysis of heterokaryons (see below), multicolor immunofluorescence and flow cytometry were performed with a FACS IV (Becton Dickinson) with a 3641-nm excitation beam for Indo-1 and a 501nm beam for fluorescein. Fluorescein emission was detected through a 530-nm band-pass filter, and Indo-1 emission was detected through a 390-nm long-pass filter by photomultiplier tubes fitted with either a 404 band-pass filter (near the maximal emission for Ca2+-bound Indo-1) or a 486-nm band-pass filter (near the maximal emission for Ca2+-free Indo-1). Basal versus elevated [Ca2+], was determined by comparison of the computed ratios of emission at these two wavelengths before and 1 min after exposure to mAb C305 (all at 37 "C).
For the heterokaryon complementation analysis (10) mutant cell lines were loaded with Indo-1 (as above, but at 6 p~ during the first incubation) and test partner cells were stained with mAb W6/32 (anti-HLA) and fluoresceinated goat anti-mouse immunoglobulin.
After extensive washing in serum-free RPMI medium, 2 X lo6 cells of each type to be fused were centrifuged together in a 24-well (16mm) plastic tissue culture dish to form a monolayer. The medium was removed and 0.3 ml of 50% (v/v) polyethylene glycol (EM Science, Cherry Hill, NJ) in serum-free RPMI medium was added gently at room temperature to the monolayer of cells. After 90 s, the monolayer was washed five times and then covered with complete medium. After 1 h at 37 "C, the sample was analyzed as described above. Heterokaryons (approximately 10-25% of the cells) were evident as Indo-l-positive fluorescein-positive cells.
DNA Transfection-DNA-mediated gene transfer into Jurkat and Jurkat-derived cells was performed by electroporation with a custombuilt Ekker-type electroplxation device (capacitance 1080 microfarads). A 300-V electric field pulse was discharged across a 0.4-cm path length cell suspension (lo7 cells/ml in 20 mM Hepes, pH 7.05, 137 mM NaCl, 5 mM KC], 0.7 mM Na2HP04, 6 mM dextrose). Selection of transfectants was begun 2 days after transfection in medium containing geneticin (GIBCO), 2 mg/ml. Saturation Binding AnnLysis-Binding studies for quantitating muscarinic receptor expression were performed with intact cells (IO6/ sample) using the muscarinic receptor antagonist [3H]quinuclidinyl benzilate ( [3H]QNB, 42 Ci/mmol, Amersham Corp.). Nonspecific binding was determined i n the presence of 10 p~ atropine (Sigma). Incubations were performed at room temperature in 50% RPMI 1640, 50% phosphate-buffered saline, 2 mM EGTA. [3H]QNB binding reached a plateau by 90 min, at which time bound activity was determined by filtering the reaction mixture through a glas fiber filter (Whatman GF/B) under vacuum. The filter was washed twice with 5 ml of the incubation buffer and once with 5 ml of ice-cold phosphatebuffered saline. Bound radioactivity was determined by liquid scintillation counting of the filter. All incubations were performed in triplicate, and the mean binding at each concentration was used in the curve fitting. Binding curves were analyzed by the Eadie-Hofstee L. Lanier, personal communication. one-site model of the LUNDON-1 Binding Isotherm Computer Program to determine the K d and number of specific binding sites.
Inositol Phosphate Measurement-Individual inositol phosphate metabolites were analyzed by quantitative anion-exchange chromatography essentially as described (1). In some experiments total inositol phosphates were measured by eluting with a single buffer (1 M ammonium acetate, 0.1 M formic acid). In such experiments cells were first incubated for 10 min with LiCl (20 mM), and the stimulations were performed in the presence of LiCl (10 mM) to inhibit dephosphorylation of IP,. For cholera toxin (CT) pretreatment, cells were incubated with the intact cholera toxin (List Biologicals, 0.1 pg/ ml) for 3 h at 37 "C (I).
Construction of pHMl-SFNeo-The plasmids pTPFneo (8) and pSVE-HM1 (20) were digested to completion with the enzymes XbaI and BamHI in standard restriction enzyme buffer. The 6.7-kilobase fragment of pTPFneo and the 1.4-kilobase genomic HMl insert of pSVE-MAR were purified by agarose gel electrophoresis and GENECLEAN (BiolOl, La Jolla, CA). The two fragments were ligated overnight under standard conditions with T4 ligase, and the plasmid pHM1-SFNeo was obtained by transformation of bacteria and plasmid preparation with CsCl gradient purification.

RESULTS
Isolation and Characterization ofJ.CaM3-The isolation of J.CaM3 was accomplished using methods that were very similar to those previously described for isolating J.CaM1 and J.CaM2 (9,lO). Briefly, a signaling-competent subclone of the wild-type Jurkat cell line was mutagenized by exposure to ethyl methane sulfonate and then selected by fluorescenceactivated cell sorting using the fluorescent Ca2+-sensitive indicator Indo-1 (21) to obtain cells that failed to elevate [Ca2+Ii upon exposure to the anti-Ti mAb C305. Cells thus obtained were next sorted for receptor-bearing cells, followed by three additional, successive cycles of paired ''Ca2+" and "receptor" sorts. The J.CaM3 clone was obtained by limiting dilution and screening by fluorimetry. Note that lectin-mediated growth inhibition was not utilized as an enrichment step in the isolation of J.CaM3, although it had been used for J.CaM1 and J.CaM2.
As with J.CaM1 and J.CaM2, J.CaM3 was found to have high levels of CD3/Ti on its surface, as demanded in the selection. Based on immunofluorescence and flow cytometry with the C305 (anti-Ti clonotypic), WT31 (anti-Ti framework determinant), and OKT3 (anti-CD3) mAbs, J.CaM3 expresses approximately 151 + 14% (S.D., n = 3) of the receptor levels found on the wild-type Jurkat cells. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of immunoprecipitated CD3/Ti proteins from J.CaM3 revealed the characteristic CD3 and Ti proteins that cannot be distinguished from those of Jurkat (data not shown). Two-dimensional isoelectric focusing analysis of immunoprecipitated proteins also revealed no gross differences in the CD3/Ti proteins (data not shown).
Despite the presence of high levels of receptor complexes recognized by available anti-CD3/Ti mAbs, J.CaM3 exhibited substantially impaired Ca2+ mobilization responses to many of these mAbs. For example, J.CaM3 demonstrated virtually no elevation of [Ca"], in response to the anti-Ti mAb R140 ( Fig. lB), which is a potent agonist for Jurkat cells (Fig. lA).
Similarly, J.CaM3 exhibited little response to the anti-CD3 mAb OKT3 (Fig. 10) in contrast to the large response of Jurkat (Fig. 1C). However, addition of a cross-linking second antibody ( i e . rabbit anti-mouse Ig) appeared to reconstitute the agonist potential of OKT3 for J.CaM3 (Fig. 1D). The responses of J.CaM3 to a large panel of mAbs are compared with those of Jurkat, J.CaM1, and J.CaM2 in Table I. This summary reveals that J.CaM3 is very similar to J.CaM1 in its responsiveness to certain anti-CD3 mAbs (235, anti-Leu4, L143, cross-linked OKT3, or C305 plus OKT3) and its lack of responsiveness to other anti-CD3 mAbs (OKT3, UCHT1, and A32.1) and to all anti-Ti mAbs examined (C305, R140, and WT31). Although the magnitude of these responses differ somewhat between the two mutants, the qualitative patterns of response are very similar. Therefore, unlike those of J.CaM2, the receptors of both J.CaM1 and J.CaM3 exhibit "leaky" defects in coupling to Ca2+ mobilization upon receptor engagement by mAbs.
Other receptor-mediated signaling events in J.CaM3 also appear to parallel the profile observed in J.CaM1. First, the production of inositol phosphate second messengers following ligand binding is significantly impaired. While Jurkat cells demonstrated a 501 f 55% (S.E.) enhancement of IP3 production above basal levels in response to C305 (t = 10 min) FIG. 1. Calcium mobilization by the J.CaM3 and parental J u r k a t cell lines. Fluorimetry with Indo-1 was performed as described under "Materials and Methods." The mAbs (final concentration 1:lOOO dilution of ascites) were added where indicated by the arrows. RaM is purified rabbit anti-mouse IgG (1 pg/ml at the first arrow, 10 pg/ml at the second arrow). Where indicated ionomycin was added (1 p~) to demonstrate intracellular Indo-1.
and a 716 f 44% increase in IP1 plus IP2 production, J.CaM3 cells demonstrated virtually no change in IP3 (110 +-3%) or IP1 plus IP2 (82 f 3%) levels, respectively. Therefore, as in J.CaM1 and J.CaM2, the signal transduction defect is proximal to IP second messenger production. Interestingly, as had been observed with J.CaM1, even the Ca2+-mobilizing mAb 235 caused only a small elevation of IP3 levels in J.CaM3 (161 f 18%) compared with the wild-type cells (769 f 64%) despite the "normal" immediate Caz+ mobilization response. As we have suggested previously, therefore, the short-term mobilization of Ca2+ in these cells requires only a small elevation of Finally, our previously described heterokaryon complementation assay made possible the rapid assignment of J.CaM3 into a known or new complementation group (10). In this assay, partner cells are alternatively loaded with the Ca2+sensitive fluorescent dye Indo-1 or stained with a fluoresceintagged mAb reactive with an irrelevant surface marker, and the partners are then fused by brief exposure to polyethylene glycol. Within 1 h, the mixture of unfused parental cells and homo-and heterokaryons are assessed on the FACS IV by three-color analysis. The Indo-1-positive, fluorescein-positive heterokaryons formed by fusion between the mutant cell line (in this case, J.CaM3) and the test partner are assayed for Ca2+ mobilization responses to C305. As summarized (Fig. 2), J.CaM3 was complemented by Ti-&deficient cells (J.RT3-T3.5 and MOLT13) and by Ti-a-deficient cells (PEER and MOLT13), but not by homokaryon (self) fusion. Therefore, as with J.CaM1 and J.CaM2, the Ti subunit is not the site of the defect in J.CaM3. Importantly, J.CaM3 was complemented by fusion with either J.CaM1 or J.CaM2, indicating that these three mutants all lie in different complementation groups (J.CaM1 and J.CaM2 were previously shown to complement one another (10)).
Introduction of H M l Receptor into Jurkat Cells-The defects in the three J.CaM mutants all appear to reside proximal to the production of IP second messengers by CD3/Ti. By examining the influence of these mutations on non-CDS/Ti receptor function we hoped to acquire additional information about the normal contribution of the mutated molecules to signal transduction. The endogenous CD2 molecule exhibits a concomitant signaling defect in J.CaM1 (ll), but CD2 also appears to depend on surface expression of CD3/Ti for signal transduction competence (11,12). CD3/Ti function in Jurkat, IPS (22).  however, does not depend on the presence of CD2 (23). Since both J.CaM2 and J.CaIM3 have spontaneously lost expression of CD2, it would be difficult to assess CD2 function in these cells. Instead, we chose to introduce a heterologous PI-coupled receptor into the parental and mutant lines and to evaluate its signaling function in these hosts. The human muscarilnic acetylcholine receptor subtype 1 is a PI-coupled member of the seven-transmembrane domain receptor family (24). For expression in the Jurkat family of cells, an expression plasmid was constructed by replacing the Ti-P cDNA in the pT@I?neo vector (8) with the genomic HM1 clone (kindly provided by D. Capon and E. Peralta, Genentech). In the resulting plasmid (Fig. 3A, pHM1-SFNeo) the HM1 gene is driven by the Friend spleen focus-forming virus long terminal repeat, and the selectable gene encoding neomycin phosphotransferase is driven by an SV40 promoter/ enhancer sequence. pHM1-SFNeo was introduced into Jurkat by electroporation and stable transfectants were selected in G418, as previously described (10). Individual clones were screened for receptor expression by assessing saturation isotherm binding of the muscarinic receptor antagonist [3H]QNB. While parental Jurkat cells demonstrated virtually no detectable [3H] QNB binding (data not shown), the representative clone J-HM1-2.2 demonstratedl significant saturable binding ("total" in Fig. 3B). Binding of [3H]QNB to J-HM1-2.2 was virtually completely inhibitable by atropine, a second muscarinic-specific antagonist ("nonspecific" in Fig. 3B). The binding kinetics from triplicate reactions for each concentration were analyzed using the microcomputer-based LUNDON-1 saturation isotherm data analysis program (the Eadie-Hofstee one-site binding model). These analyses demonstrated that J-HM1-2.2 exhibits approximately 9000 binding sites per cell, with a  but not in native Jurkat cells (data not shown).
Signaling Function of HMl in Jurkat and CD3/Ti-negative Variant-Since the HM1 receptor has been shown to exhibit coupling to the PI second messenger system in other hosts (24) we examined this function in the Jurkat transfectants. In Jurkat a very sensitive indicator of IPS production is an elevation of [Ca2+],. In Ca2+ fluorimetry studies untransfected Jurkat cells demonstrated no change in [Ca"], in response to the muscarinic agonist carbamylcholine (carbachol), but showed a substantial elevation in response to the anti-Ti mAb C305 (Fig. 4A). In contrast, J-HM1-2.2 showed a marked elevation of [Ca2+]i upon addition of carbachol, which was immediately and completely reversible with atropine (Fig.  4B); subsequent addition of C305 demonstrated the functional integrity of CD3/Ti in this cell and the failure of the heterologous muscarinic receptor to desensitize CD3/Ti, at least acutely (Fig. 4B).
Significant elevation in IP levels in J-HM1-2.2 was also seen upon exposure to carbachol (Fig. 5B). Unlike Jurkat cells (Fig. 5A), J-HM1-2.2 cells demonstrated large increases in IP3 and IP1 plus IPz metabolites as early as 2 min following addition of carbachol, and these increases were sustained for at least 20 min (Fig. 5B). Note that the induction of IP production by carbachol was similar in magnitude to that seen in response to C305.
Since the CD2 molecule is known to depend functionally on the surface coexpression of CD3/Ti, we assessed whether or not HM1 receptor function depended upon CD3/Ti expression in Jurkat. During the screening of Jurkat transfectants Inositol phosphates were extracted and analyzed as described under "Materials and Methods," with the IP1 and IP, metabolites analyzed as a single combined elution fraction and IP3 as a second fraction. Incubations were either with nothing (None), with carbachol (100 p~) , or with C305 (ascites, final dilution 1:lOOO) for the indicated lengths of time.
for HM1 expression, a positive clone was found (J-HM1-2.1) that had spontaneously lost expression of CD3/Ti from the cell surface. The absence of CD3/Ti was demonstrated both by immunofluorescence assays which revealed no detectable surface expression of receptor (data not shown) and by Ca2+ fluorimetry; we have found that ea2+ mobilization is a n extremely sensitive qualitative assay of antigen receptor expression. Clone J-HM1-2.1 showed no detectable response to the C305 (anti-Ti) or OKT3 (anti-CD3) mAbs in the fluorimeter, but exhibited a substantial response to carbachol (Fig. 4C).
Thus, CD3/Ti expression does not appear to be a prerequisite for HM1 signaling function in Jurkat-derived cells.
Partial Effects of Cholera Toxin on HMl -Since muscarinic receptors previously have been shown to utilize GTP-binding proteins (G proteins) to interact with the second messenger machinery (25,26), the above findings suggested either that the Jurkat cells natively produce the usual HM1-coupled G protein (despite their lack of endogenous HM1 receptors), or that the transfected HM1 receptor simply "borrows" a different but related endogenous G protein for its own use. It is widely predicted that the antigen receptor will also be found to utilize a G protein, but there is presently little direct evidence supporting this notion. The best evidence to date is the observation that cholera toxin, which is known to ADPribosylate and concomitantly alter the function of some G proteins (27), virtually abolishes CD3/Ti signaling function through a non-CAMP-mediated mechanism (28).
Although CT has not been observed to affect muscarinic receptor function in native hosts (29), we examined its effects in the Jurkat transfectants. Consistent with the findings of Imboden et al. (28), 3-h incubation with CT almost entirely eliminated production of IP metabolites in response to C305 (Table 11). At either saturating or subsaturating doses of C305 the production of IPS was inhibited by at least 93% following a 3-h incubation with CT. In contrast, HM1 responses to carbachol were only partially inhibited by identical CT treat- 100 p M Ascites, 1:lOOO dilution. e Ascites, 1:125,000 dilution.  (Table 11), with only 65-66% inhibition of the induction of IP production at saturating or subsaturating doses of carbachol. The virtualhy complete inhibition of CD3/Ti function and the limited inhibition of HM1 function was a consistent finding in mu1t:iple experiments. Similar 3-h incubations with the CAMP analogue dibutyryl-CAMP at doses from 1 to 500 p~ caused no inhibition of the IP responses of J-HM1-2.2 to either anti-Ti or HM1 agonists (data not shown).
HMl Function in CJD3/Ti Signaling Mutants-To aid in defining the mutated molecules in the signaling mutants J.CaM1, J.CaM2, and J.CaM3 were transfected with pHM1-SFNeo, and receptor-bearing clones were isolated as described for Jurkat. Northern analysis demonstrated significant HM1 expression in three representative transfectants derived from these mutants (data not shown), although the transfected J.CaM3 cells consistently expressed lower levels than did the other mutants. The number of HM1 receptors per cell was assessed by binding isotherm analysis with [3H]QNB, yielding: J.CaM1-HM1-1.3 = 14,000 sites/cell, J.CaM2-HM1-1.4 = 12,000 sites/cell, and J.CaM3-HM1-1A.12 = 2,000 sites/ cell.
The functional capacity of the transfected HM1 receptor was assessed by IP production, since early Ca2+ mobilization is not directly proportional to IP3 production. While endogenous CD3/Ti in all the transfected mutants failed to mediate IP production, as expected, the HM1 receptors were competent in the context of all three CD3/Ti signaling mutants (Fig. 6). Carbachol elicited 7-&fold increases in total IP production in transfected J.CaM1 and J.CaM2, and 3.5-fold increases in J.CaM3; the lower response in J.CaM3 probably is attributable to the lower receptor number, since the transfected wild-type Jurkat cells also demonstrated a quantitative dependence on receptor number (data not shown). Ca2+ mobilization responses to carbachol were similar in both transfected mutants and transfected wild-type cells, and the relative production of individual IP metabolites were comparable in the mutant and wild-type hosts (data not shown). Therefore, the mutations in the J.CaM family all affect molecules that function in an antigen receptor-specific signal transduction system and therefore have no effect on the HM1 signal transduction function.

DISCUSSION
The molecular complexity of the T cell antigen receptor complex and the lack of a cell-free reconstitution system prompted us and others to employ somatic cell genetics to explore structure/function relationships within this receptor system. Previous studies of the mutants J.CaM1 and J.CaM2 demonstrated that each has a different signaling deficit, with J.CaM1 exhibiting partial responsiveness to some anti-CD3/ Ti mAbs and J.CaM2 exhibiting a fully nonresponsive phenotype to all mAbs assessed (9, 10, 15). Complementation studies involving somatic cell hybridization, gene transfer, and a heterokaryon assay indicated that the Ti subunit is normal in both mutants (10). Moreover, J.CaM1 and J.CaM2 complement each other, suggesting that each cell line has a mutation in a different non-antigen-binding molecule involved in the CD3/Ti signal transduction process (10). We proposed that the partial integrity of responses of J.CaM1 to certain anti-CD3 mAbs and the absence of Ti-mediated signaling function implies that CD3 itself may be serve to couple Ti to other components of the signal transduction pathway.
The present studies of the new mutant J.CaM3 extend the genetic analysis of T cell antigen receptor function. Like the earlier mutants, J.CaM3 expresses high levels of grossly normal CD3/Ti complexes. Both electrophoretic analysis of im-munoprecipitated Ti and CD3 proteins and epitope studies using immunofluorescence with anti-CD3/Ti mAbs offer no clues as to the structural basis of impaired receptor function. Despite abundant levels of receptor on the cell surface, however, J.CaM3 exhibits impaired signal transduction like that of J.CaM1. First, only certain anti-CD3 mAbs elicit mobilization of intracellular Ca2+ while anti-Ti mAbs and other anti-CD3 mAbs are less effective. These features imply that the defect in signaling is relatively proximal in the pathway. Second, the failure to promote Ca2+ fluxes is attributable to the absence of coupling to IP production, also implying an "upstream" mutation. Third, those mAbs that do retain agonist function (e.g. 235) for J.CaM3 and for J.CaM1 in terms of Ca2+ mobilization appear to have only minimal agonist function with regard to IP production in the two cell lines.
The similarity of phenotypes between J.CaM1 and J.CaM3 is particularly intriguing in view of the complemention studies demonstrating that the mutations in the two cell lines lie in different complementation groups, both of which are distinct from the complementation groups defined by Ti. These findings suggest that the mutations in J.CaM1 and in J.CaM3 affect different molecules in the signaling apparatus, implying the necessity of a third non-antigen-binding molecule in the coupling of the receptor to second messenger production. While little information is available regarding the identity of the mutated molecule in J.CaM2, the "leaky" phenotypes of J.CaM1 and J.CaM3 are suggestive of early components in the pathway. The existence of mutations affecting two separate components but causing nearly indistinguishable partially permissive phenotypes is consistent with the possibility of a multimolecular complex subserving signal transduction function for the receptor. In such a model, two proteins with related functions might both be anticipated to be potential loci for mutations leading to similar functional deficits, as seen with J.CaM1 and J.CaM3. It is tempting to hypothesize that the well characterized CD3 complex represents the functional complex implicated by the genetic analyses, and that J.CaM1 and J.CaM3 will be found to have mutations in different but related CD3 chains or associated proteins. For example, in the murine T cell system there is some evidence linking expression of the CD3-{/7 dimer to the signal transduction competence of the receptor complex (30). Molecular analyses are currently in progress to test this hypothesis.
As a further tool in dissecting the signal transduction pathways, we have utilized gene transfer to introduce a heterologous PI-coupled receptor into the Jurkat-derived cell lines. Previous studies of the endogenous T cell-specific receptor CD2 demonstrated that the function of this molecule is dependent on both the expression and functional integrity of the CD3/Ti complex (11,12). Expression of the HM1 receptor in Jurkat-derived cells allowed us to explore the relationship of this receptor with that of the CD3/Ti complex. Although native Jurkat cells express no detectable HM1 receptors, the present studies indicate the availability of the appropriate machinery for HM1 function. Levels of HM1 receptor in transfected cells that are similar to those of endogenous CD3/ Ti complexes mediate comparable induction of second messenger production upon exposure to receptor agonist; the acetylcholine analogue carbachol promotes both production of IPS and elevation of [Ca2+li, as do CD3/Ti ligands. Receptor-mediated activation of second messenger production is completely and immediately reversible by atropine, a muscarinic antagonist. Furthermore, the coupling of HM1 to second messenger production in T cells does not depend on the expression of functional CD3/Ti complexes, suggesting that such dependence is a unique property of T lineage-specific receptors such as CD2 (11, 12), Thy-1 (31), Tp103 (32), and Ly6 (33).
To characterize further these signaling pathways in the T cell host we examined the influence of cholera toxin on HM1 and CD3/Ti function. Others have demonstrated that prolonged exposure of Jurkat cells to CT virtually completely inhibited CD3/Ti signaling function independently of elevations of cAMP (28). While the mechanism of inhibition of CD3/Ti function is unknown, it is possible that an uncharacterized G protein that mediates antigen receptor signaling is a substrate for CT, resulting in its debilitation. Other possible mechanisms for this inhibition include indirect effects which lead to receptor or coupling protein modification, or inositol phospholipid depletion (34). In the present studies we confirmed the complete inhibition of CD3/Ti function and observed only a partial inhibition of HM1 function. The partial impairment of HM1 function contrasts with previous studies showing the absence of CT and pertussis toxin effects in native hosts. Neither receptor system appeared to be affected by even high concentrations (500 HM) of dibutyryl-CAMP, suggesting that the CT effects on CD3/Ti and HM1 may not be mediated by the concomitant elevation of cAMP caused by G, activation. This is in contrast to some studies in which cAMP or its analogues have been shown to inhibit signal transduction by the T cell receptor in a murine T cell hybridoma (35). In Jurkat cells, however, the effects of cholera toxin cannot be attributed solely to the elevated levels of cAMP that are induced (28,36). Definitive studies to resolve these questions may require more extensive somatic cell genetics involving the CAMP-mediated kinase pathways.
The differential sensitivity of the CD3/Ti and HM1 receptor systems suggests two conclusions. First, CT-mediated inhibition of receptor function in these cells is unlikely to result from depletion of inositol phospholipid substrates or impairment of phospholipase C, since such mechanisms might be expected to influence equivalently all receptor-mediated signaling processes that utilize this second messenger system. Second, the HM1 and CD3/Ti systems both appear to involve CT-sensitive components, but these components must not be wholly identical in view of the quantitatively different effects. Whether or not the relevant CT substrates are G proteins remains to be determined. Recent work with the muscarinic receptor expressed in heterologous hosts (Chinese hamster ovary cells) suggested strongly that a cell can express multiple distinct G proteins capable of coupling to phospholipase C and that a given receptor type can utilize more than one variety of G protein in such an environment (26). Therefore, potential explanations for the partial sensitivity of HM1 to CT in Jurkat cells include: (a) use of a G protein by HM1 that is distinct from, but related to, that of CD3/Ti and that is partially CT-sensitive; and ( b ) use of two G proteins by HM1, one of which is completely resistant to CT and one of which is sensitive to it (e.g. the putative CD3/Ti G protein).
Further studies with cAMP analogues and with CAMP-dependent protein kinase mutants should help to clarify some of these important issues.
The introduction of a heterologous receptor into the J.CaM family of mutants provided an additional means to dissect the various components and pathways leading to intracellular second messenger generation. Since the mutants define a minimum of three non-Ti components of the antigen receptor signaling system, we predicted that some of the mutations might affect segments of the system that are common to other receptor-mediated signaling pathways. Based on previous work with CD2 we hypothesized that there might be a general interdependence among cell surface receptors. The experi-ments with the transfected mutants clearly demonstrated, however, that the HM1 receptor pathway is largely independent of the CD3/Ti pathway. None of the three mutants demonstrated substantially impaired signal transduction via the heterologous HM1 receptor, despite their deficiencies in CD3/Ti function. These findings imply that the mutations in these cells affect components that are used specifically by the antigen receptor, a conclusion that is consistent with the hypothesized antigen receptor-associated multimolecular signal transduction apparatus that may be altered in the mutants.
The mutant and heterologous receptor model systems should permit further exploration of the molecular interactions underlying the signal transduction function of the antigen receptor. It should also be possible to make direct comparisons of the abilities of the antigen and muscarinic receptors to mediate the necessary signals for cellular activation. Finally, molecular analysis of the known receptor components may provide insight as to the loci of the mutations in the J.CaM family and as to the role of the affected molecules in receptor function.