Genetic Analysis of Mammalian G-Protein Signalling
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Oncogene (1998) 17, 1375 ± 1381 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc 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 matura- tion. 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).