Genetic Analysis of Mammalian G-Protein Signalling

Stefan Oermanns2 and Melvin I Simon1

1Division of Biology 147-75, California Institute of Technology, Pasadena, California 91125, USA; 2Institut fuÈr Pharmakologie, Freie UniversitaÈt Berlin, Thielallee 69-73, 14195 Berlin, Germany

Heterotrimeric G-proteins are important signalling studies at the cellular or subcellular level, little is proteins which function in all cells of the mammalian known about their role in function and development of organism. Inactivating mutations in a variety of G- the whole organism. protein a-subunit genes in mice resulted in mostly The development of gene targeting techniques in unexpected phenotypes and have provided interesting mice has made possible the study of G-protein new insight into their biological roles. Whereas the signalling in the context of the whole organism and inactivation of some Ga genes led to mild phenotypes allows us to address questions regarding the biological suggesting the presence of redundant or compensatory function of individual G-proteins or G-protein families. mechanisms, other G-proteins appear to play highly The ®rst mutation of a G-protein a-subunit was speci®c biological or developmental roles. The purpose of published less than 3 years ago (Rudolph et al., this review is to summarize the current knowledge about 1995). Since then, the majority of the known Ga G-protein functions based on gene-inactivation studies. genes have been inactivated with mostly unexpected results (Table 1). As functional dierences between G- Keywords: G-protein; gene targeting; transgenic mouse; protein bg-complexes appear to be less pronounced, transmembrane signalling researchers have been cautious so far in targeting Gb or Gg genes. In this review, we will summarize and discuss the recent analyses of Ga mutant mice.

Introduction Inactivation of Gai/o family genes The functions of individual cells in multicellular organisms are regulated and orchestrated by a vast The inactivation of the Gai2 gene was the ®rst Ga array of extracellular signals including hormones, knockout published (Rudolph et al., 1995). The growth factors, paracrine factors, neurotransmitters ubiquitously expressed G-protein a-subunit Gai2 as or sensory stimuli. Cells receive many of these signals well as its close relatives Gai1 and Gai3 have been through seven transmembrane receptors which couple implicated in the receptor-dependent inhibition of to heterotrimeric guanine nucleotide binding proteins adenylyl cyclases. Since Gi-type G-proteins are usually (G-proteins). Heterotrimeric G-proteins consist of a-, present at relatively high abundance, they also b- and g-subunits and regulate the activity of various represent an important source for bg-complexes. One eector enzymes and ion channels (Hepler and Gilman, hundred per cent of Gai2 de®cient mice bred on an 129/ 1992; Neer, 1995). G-proteins are de®ned by the Sv (inbred) background eventually develop lethal, identity of their a-subunits. So far, 16 genes encoding diuse in¯ammatory bowel disease which resembles

G-protein a-subunits have been detected in mammals, in many aspects ulcerative colitis in humans. Gai2 and they appear to be involved in all aspects of de®cient mice outbred on a 129/Sv6C57BL/6 back- mammalian physiology. Some G-proteins are very ground showed greatly reduced occurrence of the specialized like those expressed speci®cally in sensory disease. Gai2 de®cient mice show increased numbers cells, others appear to have functions in a wide variety of CD4+CD87 and CD47CD8+ thymocytes with of cells and tissues. The a-subunits of mammalian G- increased staining of CD3. This altered thymocyte proteins are divided into four subfamilies based on maturation has also been described in transgenic mice homologies in sequence and function (Simon et al., speci®cally expressing the S1 subunit of pertussis toxin

1991) (Table 1). Each cell expresses multiple G- which functionally inactivates all members of the Gi- proteins, but each cell type contains a distinct set of family in thymocytes (Chan et al., 1990). However, in G-proteins. Most G-proteins couple speci®cally to addition the S1-transgenic mice show defective certain receptors and eectors, however, individual lymphocyte homing to peripheral lymphoid organs receptors and eectors can interact with more than one which was not seen in Gai2 de®cient mice. This suggests type of G-protein. Thus, some G-proteins appear to that either Gai3 or several Gi-type G-proteins, in a have overlapping distributions and functions indicating redundant fashion, mediate signalling processes in- that complex functional relationships exist among volved in T-cell homing to spleen and lymph nodes. A dierent G-proteins. While the current knowledge variety of other abnormalities were observed in Gai2 about heterotrimeric G-proteins is mainly based on de®cient mice, and in subsequent studies, dramatic changes in the phenotype and function of intestinal lymphocytes and epithelial cells have been described

(HoÈ rnquist et al., 1997). On a cellular level, Gi2 may be involved in T cell activation and thymocyte maturation. Furthermore, receptors for most chemokines have

Correspondence: MI Simon been shown to couple to Gi-type G-proteins. Thus, Genetic analysis of mammalian G-protein signalling S Offermanns and MI Simon 1376 Table 1 Mammalian G-protein a-subunits and phenotypes resulting from mutations in Ga genes Family Subtype Expression Eectors Phenotype Reference

a Gas Gas ubiquitous AC (all types): embryonic lethal, parent-of-origin speci®c defects [*] in heterozygotes Gaolf olfactory epithelium AC: no mouse mutant available ±

Gai/o Gai1 widely distributed no obvious phenotype seen so far [{] Gai2 ubiquitous AC (types I,V,VI); in¯ammatory bowel disease [1] Gai3 widely distributed no obvious phenotype seen so far [{] b 2+ 2+ Gao neuronal, neuroendocrine Ca -ch. (L-/N-type); CNS defects, defective Ca -ch. regulation in the [2,{] heart Gaz neuronal, platelets AC; ? viable, defective platelet activation [{] Gagust taste cells, brush cells ? impaired bitter and sweet sensation [3] Gat-r retinal rods, taste cells cGMP PDE: no mouse mutant available ± Gat-c retinal cones cGMP PDE: no mouse mutant available ±

Gaq Gaq ubiquitous cerebellar ataxia, defective platelet activation [4] Ga11 almost ubiquitous PLC-b: no obvious phenotype seen so far [}] Ga14 kidney, lung, spleen (b35b144b2;b4) no obvious phenotype seen so far [$] c Ga15 hematopoietic cells no obvious phenotype seen so far [}]

Ga12 Ga12 ubiquitous ? no mouse mutant available ± Ga13 ubiquitous ? defective embryonic angiogenesis (lethal) [5] a b c Four splice variants, Two splice variants, mouse form (Ga16, human counterpart). *L Weinstein (personal communication) {L Birnbaumer, M Jiang, G Boulay, K Spicher (personal communication); {IA Hendry (personal communication); }T Wilkie (personal communication); $Huiping Jiang and MIS (unpublished observation); 1Rudolph et al., 1995; 2Valenzuela et al., 1997; 3Wong et al., 1996; 4Oermanns et al., 1997b and in press; 5Oermans et al., 1997a

while there are a variety of possible mechanisms have occasional seizures, which could be due to subtle

whereby inactivation of the Gai2 gene may cause a alterations in the architecture of the central nervous defect in the regulation of immune responses eventually system or to some other functional defects. In addition, leading to the development of in¯ammatory bowel severely abnormal motor behaviour can be observed in

disease, the exact pathogenic process is still obscure. Gao de®cient mice (L Birnbaumer, M Jiang, G Boulay, K t is also not clear to what extent a lack of the Spicher; personal communication). Severe functional

biological Gai2 function can be compensated for by nervous system defects in mice lacking Gao would be

other Gai subtypes (e.g. Gai1 and Gai3). Studies in predicted based on the general view that Go couples

primary cells derived from Gai2 de®cient mice as well as receptors for various neurotransmitters to the inhibitory 2+ in Gai2 (7/7) embryonic stem cells suggest that regulation of neuronal Ca channels (Schultz et al., 1990; inhibitory regulation of adenylyl cyclase through Kleuss et al., 1991; Wickman and Clapham, 1995). The

various receptors in the absence of Gai2 is at least neurological defects observed in Gao de®cient mice

partially mediated by Gai1/Gai3 (Raymond et al., 1994; demonstrate that the lack of a high abundance signalling Rudolph et al., 1996). In embryonic stem cell derived protein does not necessarily predict that its function is

cardiocytes both Gai2 and Gai3 are necessary for absolutely essential. In contrast to its abundance in the

receptor mediated regulation of IK+Ach (Sowell et al., central nervous system, Gao represents only a minor

1997). In contrast to Gai2 de®cient mice, mice lacking fraction of all G-proteins in the heart. Nevertheless, it was

Gai1 or Gai3 do not seem to have obvious phenotypic shown that the inhibitory muscarinic regulation of changes (L Birnbaumer, M Jiang, G Boulay, K cardiac L-type Ca2+ channels in the heart was completely

Spicher; personal communication). Crosses of differ- abrogated in the Gao (7/7) mice (Valenzuela et al.,

ent Gai mutant mice will help to elucidate the 1997), indicating that inhibitory muscarinic regulation of functional relationships among the closely related cardiac L-type Ca2+ channels is speci®cally mediated by

members of the Gai family. Go. However, since wild-type and Gao de®cient mice were

The G-protein Go is expressed in neuronal and indistinguishable upon electrocardiographic analysis, the 2+ neuroendocrine cells as well as at low levels in the heart. physiological signi®cance of Go mediated Ca channel It is the most abundant G-protein in the mammalian inhibition in the heart is not clear. Certainly, the

nervous system; in growth cone membranes, Go makes up inactivation of the Gao gene in mice has so far

10% of the membrane protein (Strittmatter et al., 1990). demonstrated that Gao serves speci®c functions in the

Since the activity of Gao can be regulated by GAP43, heart as well as in the nervous system which are obviously another protein enriched in growth cones and involved in not taken over by other G-protein a-subunits.

neuronal path®nding (Strittmatter et al., 1990, 1995), Gao The sensory G-proteins Golf, rod-transducin, cone- has been suggested to be important for neuronal transducin and gustducin are expressed at detectable

guidance. Surprisingly, Gao (7/7) mice showed no levels only in specialized sensory cells. No mutants are

gross morphological abnormalities, and neuronal path- available for Golf, a member of the Gs family, and ®nding as well as growth cone collapse appeared to be transducins. Their functions appear to be obvious normal in these animals (Valenzuela et al., 1997). given their expression together with odorant receptors

However, Gao de®cient mice appear to be handicapped and the light receptors rhodopsin and opsins in the and have greatly reduced postnatal survival rate with 50% respective highly specialized sensory cells. However,

of the animals dying a few weeks after birth. Gao de®cient gustducin-de®cient mice have shown that surprises are mice have been reported to suer from tremors and to possible (Wong et al., 1996). Among the four taste Genetic analysis of mammalian G-protein signalling S Offermanns and MI Simon 1377 qualities sweet, bitter, sour and salty, bitter and sweet primarily responsible for coupling of receptors in a tastes are believed to be transduced through hetero- pertussis toxin insensitive manner to phospholipase C trimeric G-proteins. While sweet sensation seems to b-isoforms (Strathmann and Simon 1991; Exton, 1996). involve the activation of adenylyl cyclase in taste Receptors activating Gq family members in mammalian receptor cells, bitter substances lead to the stimulation systems do not discriminate between Gaq and Ga11 of phospholipase C, and there is also evidence that (Wange et al., 1991; Wu et al., 1992; Oermanns et al., bitter tastants regulate phosphodiesterase activity. 1994a). Similarly, there appears to be little dierence Gustducin is a G-protein only expressed in taste cells between the abilities of both G-protein a-subunits to and some chemoreceptor cells of the intestine regulate phospholipase C b-isoforms; whereas Gaq and

(McLaughlin et al., 1992; HoÈ fer et al., 1996). It has Ga11 indistinguishably activate the b1, b3 and b4 high homology to transducins which regulate cGMP- isoforms of PLC, both are equally poor regulators of phosphodiesterase activity in retinal photoreceptor PLC b2 (Blank et al., 1991; Wu et al., 1992; Hepler et cells. Therefore, gustducin has been proposed to al., 1993; Jiang et al., 1994; Lee et al., 1994). In couple receptors for bitter tastants to speci®c taste contrast to Gaq and Ga11, the murine G-protein a- receptor phosphodiesterases. As predicted, gustducin- subunit Ga15 and its human counterpart Ga16 are only de®cient mice showed impaired electrophysiological expressed in a subset of hematopoietic cells, and the and behavioural responses to bitter agents while expression of Ga14 is restricted to several organs e.g. responses of gustducin (7/7) mice to sour and salty kidney, testis and lung (Amatruda et al., 1991; Wilkie stimuli were indistinguishable from those of wild-type et al., 1991). Despite the speci®c expression patterns of mice (Wong et al., 1996). Surprisingly, behavioural and Ga14 and Ga15/16 which may point to cell type speci®c neuronal taste sensitivity to sweet tastants was also functions no obvious phenotypic changes were impaired, suggesting that gustducin is involved in the observed in mice carrying inactivating mutations of sweet and bitter signalling in taste receptor cells the Ga14 and Ga15 genes (H Jiang, MIS and T Wilkie; (Lindemann, 1996; Kinnamon and Margolski, 1996). unpublished data). Their functions may be dispensable

or compensated for by other members of the Gaq family. It is also possible that the defects resulting from Null mutations in Gaq family genes loss of their functions are subtle and have evaded analysis so far.

Members of the mammalian Gaq family are involved in Mice with a null mutation in the Gaq gene are born the pertussis toxin insensitive regulation of phospholi- alive but develop an ataxia with clear signs of motor pase C b-isoforms (for review see Exton, 1996). The Gq coordination de®cits (Oermanns et al., in press). The family consists of four members whose a-subunits are morphology of the peripheral and central nervous expressed by individual genes with dierent expression system and in particular of the cerebellar cortex is not patterns. The biological signi®cance of the diversity overtly disturbed. However, functional defects which among the Gaq gene family which evolved by gene are most likely responsible for the ataxia in Gaq (7/7) duplication in higher multicellular organisms (Wilkie mice could be observed in the cerebellar cortex (Figure and Yokoyama, 1994) is currently not clear. Gaq and 1). While excitatory synaptic transmission from parallel

Ga11 appear to be expressed ubiquitously and are ®bers (PFs) to cerebellar Purkinje cells (PCs) and from

Figure 1 Main neuronal connections of the cerebellar cortex and some of the putative mechanisms of cerebellar LTD. The cerebellar cortex has basically one output, the Purkinje cell (PC) axon and two inputs, one in form of climbing ®bers (CFs) which directly synapse on PCs and one in form of Mossy ®bers (MFs) which synapse on granule cells (GCs). GCs send their bifurcating axon (parallel ®bers) into the outer layer of the cerebellar cortex where they form multiple synaptic contacts with PCs. Long-term depression (LTD) of the parallel ®ber (PF)-PC synapse requires conjunctive stimulation of PFs and CFs innervating the same PC. It results from a long-lasting depression of the AMPA receptor-mediated current which is induced by an increase in the Ca2+ concentration in PC dendrites as well as the activation of metabotropic glutamate type 1 receptors (mGluR1) which signal through Gq. PLC, phospholipase C; IP3, inositol-1,4,5-trisphosphate; DAG, diacyl glycerol; PKC, protein kinase C; AMPA-R., ionotropic non-NMDA glutamate receptor Genetic analysis of mammalian G-protein signalling S Offermanns and MI Simon 1378 climbing ®bers (CFs) to PCs was functional, about in the postnatal elimination of multiple CF innervation

40% of adult Gaq-de®cient PCs remained multiply as well as in cerebellar LTD (Figure 1). It has been

innervated by CFs due to a defect in regression of shown that most cells express Gaq together with its

supernumerary CFs in the third postnatal week. In the close structural and functional homologue Ga11 cerebellum of newborn rats and mice, each PC is (Strathmann and Simon, 1990). In situ hybridization

innervated by multiple CFs. Massive elimination of results indicate that cerebellar PCs express both Gaq

supernumerary CF-PC synapses occurs during the ®rst and Ga11. However, Western blot analysis of cerebellar three postnatal weeks, and a state is reached at about membranes and immunohistochemistry of wild-type

postnatal day 20 in which most PCs are innervated by and Gaq de®cient cerebellum indicate that the levels of

single CFs. This developmental change which is a Gaq found in the cerebellar cortex clearly exceeded

prerequisite for proper function of the cerebellar cortex those of Ga11 by several fold. It is, therefore, concluded

appears to depend on the presence of intact granule that PCs predominantly express Gaq, and that the

cells and of functioning synapses between PFs and PCs remaining Ga11 is not able to compensate the lack of

(Conquet et al., 1994; CreÂ pel, 1982; Kano et al., 1995). Gaq. Accordingly, Ga11 de®cient animals do not show A defect in the modulation of the PF-PC synapse in any signs of motor discoordination (T Wilkie, personal

mice lacking Gaq is also suggested by the fact that long communication). However, one cannot rule out the

term depression (LTD) of the PF-PC synapse was possibility that Gaq and Ga11 have dierent subcellular

de®cient in Gaq (7/7) mice. Cerebellar LTD is a form localizations within cerebellar PCs and that mGluR1

of synaptic plasticity believed to be a cellular substrate dierentially interacts with Gaq and Ga11.

for some forms of motor learning and to involve the While most mammalian cells express Gaq together

modulatory regulation of excitatory AMPA receptors with its close structural and functional relative Ga11, at the PF-PC synapse (Ito, 1989; Linden, 1994) (Figure platelets are an exception in that they contain only

1). Very similar phenotypes have been described for Gaq. The main physiological stimuli activate platelets mice lacking the metabotropic glutamate type 1 through G-protein coupled receptors which are able in

receptor (mGluR1) (Aiba et al., 1994; Kano et al., turn to activate Gq,Gi and G12/13 (Shenker et al., 1991;

1997) which couples to Gaq/11 as well as for mice Hung et al., 1992; Oermanns et al., 1994b; Ushikubi

de®cient in the b4 isoform of phospholipase C which et al., 1994) (Figure 2). Gaq de®cient platelets did not shows predominant expression in cerebellar Purkinje aggregate and secrete their granule contents in response cells (Roustan et al., 1995) (H Jiang, M Kano, MIS, D to a variety of physiological platelet stimuli including

Wu; unpublished observation). The mGluR1, Gaq and thrombin, collagen, ADP and the thromboxane A2 PLCb4 are colocalized in dendritic spines of PCs, mimetic U46619 (Oermanns et al., 1997b). The

suggesting that a de®ned signalling cascade in the unresponsiveness of Gaq-de®cient platelets in vitro

postsynaptic membrane of PF-PC synapses is involved correlated with a hemorrhagic diathesis of Gaq

Figure 2 Role of Gq in platelet activation. The main physiological platelet activators are collagen, thrombin, thromboxane A2 (TXA2) and ADP which directly or indirectly function through G-protein mediated processes. Gq-mediated regulation of b-isoforms of phospholipase C appears to be the predominant pathway leading to full platelet activation which involves activation of ®brinogen receptors as well as degranulation. Platelet shape change, the spherization of platelets accompanied by the extrusion of pseudopodia, can still be induced in the absence of Gq indicating a regulation through other G-proteins (e.g. G1 and/or G12/13, PLC, phospholipase C; IP3, inositol-1,4,5-trisphosphate; DAG, diacyl glycerol; PKC, protein kinase C; PLA2, phospholipase A2 Genetic analysis of mammalian G-protein signalling S Offermanns and MI Simon 1379

(7/7) mice in vivo. In addition, Gaq-de®cient mice a-subunit with identical function and wide expression were protected from thromboembolism induced by pattern has been described. Thus, as one would predict platelet activation in vivo through intravenous injection for a non-redundant, ubiquitously expressed gene of a collagen/epinephrine mixture. Interestingly, plate- whose gene product mediates a fundamental signalling lets from a patient suering from mucocutaneous process, lack of Gas in mice results in embryonic bleeding diathesis have been shown to contain reduced lethality (L Weinstein, personal communication). The levels of Gaq (Gabbeta et al., 1997). These data clearly human autosomal dominant disease Albright heredi- showed that the Gq mediated activation of PLC is tary osteodystrophy (AHO) has been shown to result essential for induction of full platelet activation by from germ line heterozygous loss-of-function mutations physiological platelet activators and that the lack of in the Gas gene (Weinstein and Shenker, 1993; Spiegel,

Gaq cannot be compensated for by a or bg subunits of 1995). In accordance with the function of Gas, patients the other heterotrimeric G-proteins present in platelets, suer from generalized resistance to several hormones e.g. G12,G13,Gi2 or Gi3 (see Figure 2). which act through Gs-coupled receptors (e.g. parathyr-

In contrast to Gaq-de®cient mice, homozygous oid hormone, thyroid stimulating hormone, gonado- mutant Ga11 mice are normal (T Wilkie, manuscript tropins). However, it is not clear how a heterozygous in preparation). Gaq is obviously able to compensate loss-of-function mutation in the Gas gene leads to for the loss of Ga11 function, whereas Ga11 cannot complete resistance to some but not all hormones replace Gaq. A wider expression pattern of Gaq (e.g. in acting through Gs. Similarly, there appears to be some platelets), higher cellular levels of Gaq compared with phenotypic variability among patients harboring the

Ga11 in some tissues like the central nervous system or same Gas mutation. Heterozygous Gas mutant mice incompletely overlapping cellular functions or localiza- which show pleiotropic and parent-of-origin speci®c tions of both proteins may account for the observed manifestations (L Weinstein, personal communication) lack of complete functional redundancy of Gaq and represent a useful model to study the pathogenesis of

Ga11 in vivo. To elucidate the levels and degrees of AHO. redundancy of Gaq and Ga11 functions, mice carrying a Whereas considerable knowledge exists about the targeted mutation in the Gaq gene were crossed with cellular function of most members of the Gaq,Gai/o

Ga11 mutant mice (SO, MIS and T Wilkie, unpublished and Gas families, the cellular and biological function data). Analysis of the ospring from intercrosses of of G12 and G13 (which constitute the G12 family) are double heterozygous animals [Gaq(7/+); Ga11 (7/+)] not known (Dhanasekaran and Dermott, 1996). Both revealed that double homozygous mutant mice [Gaq G-protein a-subunits appear to be expressed ubiqui-

(7/7); Ga11 (7/7)] apparently develop normally until tously (Strathmann and Simon, 1991). Studies using embryonic day 9.5 (e9.5). Thereafter, they are retarded constitutively activated forms of Ga12 and Ga13 have and die at about embryonic day 11. Gaq/Ga11 double provided evidence that these G-proteins are able to de®cient embryos show a thinning of the myocardial transform ®broblasts (Xu et al., 1993; Jiang et al., layer of the heart which is already visible at e 9.5 and 1993). On a cellular level, they may be involved in the which most likely leads to embryonic death due to regulation of the Jun-kinase/stress-activated protein + + cardiac failure, indicating that both, Gaq and Ga11, may kinase pathway (Vara Prasad et al., 1995), the Na /H be necessary for proper growth of myocardial cells exchanger and the Rho-dependent formation of stress during embryogenesis. Several stimuli which activate ®bers (Voyno-Yasenetskaya et al., 1994; Dhanasekaran

Gq/G11 coupled receptors are thought to be involved in et al., 1994; Buhl et al., 1995; Hooley et al., 1996). A myocardial hypertrophy (Chien et al., 1993; Cooper, role of Ga12/13 in the regulation of cytoskeletal 1997) and transgenic expression of a constitutively functions is also supported by the ®nding that cta,a activated mutant of the a1 adrenergic receptor as well Drosophila gene closely related to Ga12 and Ga13,is as of the wild type form of Gaq in the heart has been involved in ventral furrow formation during Droso- shown to result in cardiac hypertrophy (Milano et al., phila gastrulation which is initiated by the constriction

1994; D'Angelo et al., 1997). Thus, Gaq and Ga11 of ventral furrow cells (Park and Wieschaus, 1991; appear to be involved in physiological as well as Sweeton et al., 1991). Lack of Ga13 in mice results in pathological growth of cardiomyocytes. Mice with one embryonic lethality at about embryonic day 9.5 when wild-type allele of either the Gaq gene [Gaq(7/+); gastrulation is already completed. At this stage, mouse

Ga11(7/7)] or the Ga11 gene [Gaq(7/7); Ga11(7/+)] embryos express both, Ga12 and Ga13, ubiquitously. are born alive. However, almost all of these mice die Analysis of Ga13 de®cient mouse embryos revealed that within a few hours after birth. The exact cause of this loss of Ga13 leads to a defective organization of the postnatal mortality is not clear yet. These ®ndings vascular system which is most prominent in the yolk illustrate the redundancy of Gaq and Ga11 gene sac and in the head mesenchyme (Oermanns et al., functions and demonstrate that small changes in the 1997a). Blood vessels of the head mesenchyme are dose of their genes can aect common functions of Ga1 believed to develop through angiogenetic processes and Ga11. which include sprouting, growth, migration and remodelling of existing endothelial cells (Risau, 1997).

Fibroblast-like cells obtained from Ga13 (7/7) Inactivation of Gas and Ga12 family genes embryos showed mitogenic responses to various stimuli which were indistinguishable from those of Direct stimulatory regulation of adenylyl cyclases cells cultured from wild-type embryos. However, through G-protein coupled receptors involves the G- chemokinetic eects of thrombin were completely protein Gs which is expressed ubiquitously. The Gas abrogated in cells lacking Ga13, indicating that Ga13 gene gives rise to four splice variants which appear to is required for full migratory responses of cells to be functionally indistinguishable. No other G-protein certain stimuli. It is tempting to speculate that a failure Genetic analysis of mammalian G-protein signalling S Offermanns and MI Simon 1380 of local cell movement and orientation in response to which exhibit redundant, additive or opposite functions speci®c extracellular stimuli is a cellular mechanism may contribute to many regulatory events, and this underlying the observed vascular system defects in possibility may not always be revealed in single

Ga13 (7/7) embryos. Angiogenesis requires endothe- knockouts. As shown for members of the Gaq-family, lial cell interaction with extracellular surfaces, local cell functionally related Ga genes appear to have at least movement and remodelling, processes which might not partially redundant functions which strictly depend on only be regulated through receptor tyrosine kinases a de®ned gene dosage. but also via G-protein mediated pathways. Since The next generation of mutations will include subtle

defects observed in Ga13 de®cient embryos and cells mutations and time and tissue speci®c as well as

occurred in the presence of Ga12, it has to be inducible knock-outs. Conditional inactivation of more

concluded that Ga13 and its closest relative, Ga12, than one Ga gene will allow us to study the role of G- ful®l at least partially non-overlapping cellular and proteins in speci®c signalling processes at a much biological functions. higher level of complexity. With the generation of increasingly sophisticated gene targeting techniques, we should expect exciting new insights into the biology of Conclusions and prospect G-protein mediated signalling.

The generation of mice lacking distinct G-protein a- subunits has generated a wealth of new information. In many cases completely unexpected biological functions Acknowledgements were revealed. However, ablation of a single Ga gene We thank L Birnbaumer, IA Hendry, L Weinstein and T in the germ line of mice will give us a deep but Wilkie for generously providing us with unpublished data necessarily incomplete view into the biological role of and G Schultz for critical reading of the manuscript. its gene product. Combinations of dierent G-proteins

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