©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel G Protein Subunit Containing Atypical Guanine Nucleotide-binding Domains Is Differentially Expressed in a Molluscan Nervous System (*)

(Received for publication, April 14, 1995; and in revised form, June 8, 1995)

Jaco C. Knol Arno R. van der Slik Ellen R. van Kesteren (1) Rudi J. Planta Harm van Heerikhuizen (§) Erno Vreugdenhil

From theGraduate School of Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Faculty of Chemistry, Department of Biochemistry and Molecular Biology and Faculty of Biology, Department of Experimental Zoology, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We describe the characterization of a novel G protein alpha subunit, Galpha(a). cDNA encoding this subunit was cloned from the central nervous system of the mollusc Lymnaea stagnalis. The deduced protein contains all characteristic guanine nucleotide-binding domains of Galpha subunits but shares only a limited degree of overall sequence identity with known subtypes (30%). Moreover, two of the nucleotide-binding domains exhibit salient deviations from corresponding sequences in other G protein alpha subunits. The A domain, determining kinetic features of the GTPase cycle, contains a markedly unique amino acid sequence (ILIIGGPGAGK). In addition, the C domain is also clearly distinct (DVAGQRSL). The presence of a leucine in this motif, instead of glutamic acid, has important implications for hypotheses concerning the GTPase mechanism. In contrast to other Galpha subtypes, Galpha(a) has no appropriate N-terminal residues that could be acylated. It does contain the strictly conserved arginine residue that serves as a cholera toxin substrate in Galpha(s) and Galpha(t) but lacks a site for ADP-ribosylation by pertussis toxin. In situ hybridization experiments indicate that Galpha(a)-encoding mRNA is expressed in a limited subpopulation of neurons within the Lymnaea brain. These data suggest that Galpha(a) defines a separate class of G proteins with cell type-specific functions.


INTRODUCTION

Heterotrimeric G proteins (Galphabeta) form a family of molecular go-betweens that couple stimulus-triggered cell surface receptors to response-generating effectors within the cell(1, 2, 3) . In reconstitution systems, specific G protein subtypes can interact with more than one receptor or effector subtype(1) . Such a promiscuity might, in fact, allow the integration or distribution of extracellular signals in vivo(4) . To address this issue, one needs to study intact cells since their complex plasma membrane organization furnishes a specificity of signaling that is significantly higher than that of reconstituted systems(5) . Moreover, since signaling pathways may differ considerably in different cells, it is crucial to work with unambiguously identified cell types.

One system offering such opportunities is the simple central nervous system of the pond snail, Lymnaea stagnalis, which we use to study the function of G protein-mediated signaling networks in neuronal information processing. The Lymnaea brain contains large neurons that can be reproducibly identified from animal to animal, manipulated in situ, and cultured and studied in vitro(6, 7) . Previously, we have cloned a diverse set of snail Galpha subunits, i.e. Galpha(o), Galpha(i), Galpha(s), and Galpha(q), as well as a Gbeta subunit (8, 9, 10) . All of these Lymnaea subunits appear to be remarkably similar to their mammalian counterparts (76-82% amino acid sequence identity). Taking into consideration such a striking resemblance and the fact that at least 16 Galpha subtypes exist in mammals (forming four classes), it seemed reasonable to assume that Lymnaea expresses more than merely four G protein alpha subunits (belonging to three classes).

Here, we describe the cloning of a novel Galpha subtype, Galpha(a), which has hitherto not been reported in any other species. Although clearly a G protein alpha subunit, it is unlike subtypes described to date. Two of its nucleotide-binding domains deviate markedly from analogous sequences in other Galpha proteins. These differences will probably have significant functional consequences. In situ hybridization shows that the Galpha(a) gene is specifically expressed in a minority of neurons within the Lymnaea central nervous system, suggesting that this atypical G protein subunit has a cell-specific function. Our findings indicate that the G protein family is yet more elaborate, implying that additional members remain to be discovered.


MATERIALS AND METHODS

Animals

L. stagnalis were bred under standard laboratory conditions and fed lettuce ad libitum. Adult specimens were used (shell height, 25-35 mm).

PCR^1Cloning

Total RNA was isolated from Lymnaea central nervous systems (11) and reverse-transcribed with oligo(dT) as a primer using Moloney murine leukemia virus reverse transcriptase according to the manufacturer (Life Technologies, Inc.). Degenerate PCR primers were based on conserved amino acid motifs DVGGQ and FLNKKD of Galpha domains C and G, respectively. The sense primer was as follows: GAL5, 5`-AGAGAATTCGA(T/C)GTIGGIGGICA-3` (EcoRI cloning site in italics, I = inosine). Antisense primers included GAL6, 5`-CACGGATCC(A/G)TC(T/C)TT(T/C)TT(A/G)TT(G/A/T/C)AG(A/G)AA-3` and GAL7, 5`-CACGGATCC(A/G)TC(T/C)TT(T/C)TT(A/G)TT(T/C)AA(A/G) AA-3` (BamHI cloning site in italics). The antisense primer was synthesized as two separate pools of oligonucleotides to reduce redundancy(12) .

PCR was carried out with one-tenth of an animal equivalent of cDNA and 10 µg/ml each of GAL5 and either GAL6 or GAL7 (in 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl(2), 0.01% (w/v) gelatin, 0.1% Triton X-100, 200 µM of each dNTP, 0.2 units of Super Taq polymerase (HT Biotechnology); 40 cycles of 1 min at 94 °C, 1 min at 40 °C, and 1 min at 72 °C). Under these conditions, the GAL5-GAL6 combination yielded, among others, a product of an appropriate size ( 240 base pairs), whereas the GAL5-GAL7 combination did not. The GAL5-GAL6 product was subcloned using standard procedures(13) . Inserts were sequenced with vector-specific primers using Sequenase 2.0 according to the manufacturer's instructions (U. S. Biochemical Corp.).

Isolation of cDNA Clones Encoding Galpha

A Lymnaea central nervous system-specific cDNA library (constructed in UniZAP XR (Stratagene); 29 subpools of approximately 50,000 independent clones) (^2)was screened in a PCR-based procedure(14) . Clone-specific primers (GAA1, antisense, 5`-TCATCATACGCGGTCAAGTTGAC-3`; GAA2, sense, 5`-GTCAACTTGACCGCGTATGATGA-3`) were used in combination with vector-specific primers to screen all library subpools. Selected cDNA clones were isolated by screening approximately 75,000 plaques of the pertinent subpool in a PCR-based strategy(15) . The R408a helper phage was used in the in vivo excision procedure described by the supplier (Stratagene) to recover the cDNA inserts in phagemids.

Characterization of GAAL1 and GAAL2 cDNAs

Both strands of cDNAs were sequenced with Sequenase 2.0, applying a walking primer strategy. Ambiguous regions in the sequence were re-examined by cycle sequencing with Super Taq using a SequiTherm kit (Epicentre Technologies) according to the manufacturer's instructions.

In Situ Hybridization

The insert of pGAAL2 was PCR-amplified with vector-specific primers containing T3 and T7 RNA polymerase promoter sequences, respectively. Digoxigenin-labeled cRNA probes were synthesized using a commercial kit (Boehringer Mannheim) and hybridized with 7-µm sections of the Lymnaea central nervous system(9) .


RESULTS

Identification of a Novel G Protein alpha Subunit Expressed in Lymnaea

Degenerate oligonucleotides were based on well conserved Galpha amino acid motifs within domains C and G of the nucleotide-binding pocket ((16) ; see ``Materials and Methods''). Using these oligonucleotides as primers, PCR was performed with Lymnaea central nervous system-specific cDNA as a template. A PCR product of the appropriate size was subcloned, and the insert of multiple clones was characterized by nucleotide sequence analysis.

In a collection of 34 clones analyzed, we encountered multiple known Lymnaea cDNA sequences, coding for Galpha(o) (8 clones), Galpha(i) (5 clones), or Galpha(q) (3 clones). The ratio of these clones is roughly similar to their relative abundance in the Lymnaea brain (data not shown). The other 18 clones, however, contained an identical and novel sequence. Upon conceptual translation it appeared to encode (part of) a G protein alpha subunit, as deduced from the presence of hallmark amino acid motifs directly flanking the residues encoded by the PCR primers. The predicted amino acid sequence was clearly different from that of all Lymnaea Galpha subunits characterized by us before(8, 10) . Moreover, it did not resemble any other Galpha sequence described to date.

Isolation and Characterization of cDNA Clones Encoding Galpha

To isolate cDNA clones harboring the full protein-coding region for the newly identified Galpha subunit, we screened a Lymnaea central nervous system-specific cDNA library in a PCR-based strategy(14, 15) . At least 25 out of approximately 1.5 10^6 independent clones appeared to be Galpha(a)-specific. Two of these were isolated for further analysis and designated GAAL1 (G-Alpha Ajax of Lymnaea) and GAAL2, respectively (Fig.1).


Figure 1: Schematic representation and restriction sites of the GAAL1 and GAAL2 cDNAs. Openboxes represent protein-coding sequences; straightlines represent untranslated sequences, and the wavyline indicates retained intron sequences within the GAAL 1 cDNA. B, BamHI; C, ClaI; E, EcoRI; N, NcoI; P, PstI; S, SmaI.



Partial nucleotide sequence analysis revealed that the GAAL1 insert (3.5 kilobases) corresponds to an immature RNA transcript, as it starts with (part of) a retained intron within the domain A-encoding part (Fig.2A). The 3`-intron-exon boundary follows a sequence of highly repetitive nature and exhibits a sequence (cag/GT) that complies to the consensus for splice acceptor sequences (17) . The position of the intron is identical to that of intron 1 in Galpha(i) and Galpha(s) class genes as well as Galpha genes of Caenorhabditis elegans(18, 19, 20) . In general, this intron tends to be large. Indeed, as pGAAL1 misses the 5`-part of the intron, the length of the latter should exceed 0.5 kilobases (Fig.2A).


Figure 2: Characterization of Galpha(a)-encoding cDNAs. A, partial nucleotide sequence of the GAAL1 cDNA. The 5`-part of GAAL1, harboring the downstream part of a putative retained intron and the 3`-intron-exon boundary, was sequenced. Intron sequences are given in lowercase letters, and exon sequences and the encoded amino acids are given in upper case letters. B, nucleotide sequence of the GAAL2 cDNA and conceptual translation. The regions that were used to devise degenerate PCR primers GAL5 (sense) and GAL6 or GAL7 (antisense) are overlined. Triangles indicate the position of the intron in the GAAL1 cDNA, and an asterisk denotes the stop codon. The arginine residue that is conserved in all G protein alpha subunits (Arg-212 in Galpha(a)) and serves as a cholera toxin substrate in Galpha(s) and Galpha(t) has been circled. Numbering of the nucleotide sequence is relative to the start codon.



The GAAL2 cDNA does contain a complete open reading frame. Fig.2B shows the nucleotide sequence and conceptual translation of the 1541-base pair insert. No bona fide polyadenylation signal can be found at the 3`-end of GAAL2, which probably arose from internal priming by the oligo(dT) primer used for reverse transcription. Since the GAAL1 cDNA contains approximately 1.9 kilobases of trailer sequences and open reading frame analysis of GAAL2 revealed a putative coding region of 1158 base pairs, the corresponding mRNA could be as long as 3 kilobases provided that alternative splicing does not occur. The GAAL2 cDNA specifies a 386-amino acid protein with a theoretical molecular weight of 44,666. The reading frame starts with a methionine codon in an appropriate sequence context for translation initiation(21) . Within the deduced protein sequence, domains A, C, G, and I of the putative guanine nucleotide-pocket can be easily discerned ((16) ; see also Fig.3). The novel G protein alpha subunit was designated Galpha(a).


Figure 3: Comparison of Galpha(a) with other Lymnaea G protein alpha subunits. Alignment of the amino acid sequence of the Galpha(a) protein with that of Lymnaea Galpha(i), Galpha(o), and Galpha(s)(8) as well as Lymnaea Galpha(q)(10) is shown. Numbers are relative to the Galpha(a) sequence. Guanine nucleotide-binding motifs A, C, G, and I (16) are indicated by thickoverlining. The helical domain (24) has been indicated by singleoverlining, whereas linkers L1 and L2, which connect the helical domain to the rest of the protein, are indicated by dottedoverlining. Asterisks indicate identical amino acid residues, dots indicate conserved substitutions, and dashes indicate gaps to allow for optimal alignment. Amino acid residues that appear to be conserved in G protein alpha subunits are indicated by trianglesunderneath the compared sequences.



Comparison of Galpha with Other Galpha Subunits

Fig.3shows an alignment of the predicted amino acid sequence of Galpha(a) with that of previously described Lymnaea G protein alpha subunits(8, 10) . The primary structure of Galpha(a) turns out to be only slightly similar to these proteins. In fact, it exhibits a low degree of amino acid sequence identity (27-33%) with any Galpha subtype described to date, ranging from vertebrate and invertebrate members of the Galpha(i), Galpha(s), Galpha(q), and Galpha subclasses to putatively species-specific subunits like Galpha(f) and concertina of Drosophila(22, 23) and the GPA proteins of C. elegans(20) . In sharp contrast, previously identified Lymnaea Galpha proteins share a very high degree of sequence identity with their respective vertebrate and invertebrate counterparts (76-90%; (8) and (10) ). Amino acid identity percentages such as observed for Galpha(a) generally indicate that the compared proteins are members of different Galpha subclasses. Therefore, we consider Galpha(a) to represent a novel subtype, defining a distinct class of G protein alpha subunits by itself.

Galpha(a) is most distinct in the so-called helical domain (24) , encompassing amino acids 54-218. This domain is the most heterogenous part of G protein alpha subunits and includes a region of structural disparity between Galpha(t) and Galpha(i)1, which might be related to their interaction with different proteins (25) . The Galpha(a) helical domain appears to bear an insert when compared with other Galpha proteins (Fig.3).

Different as Galpha(a) may be, it does contain 41 out of 63 residues that are conserved between members of the Galpha(s), Galpha(i), Galpha(q), and Galpha classes (not shown). When taking into account the Galpha subunits of the slime mold Dictyostelium(26) and the Galpha(s) protein of the trematode Schistosoma mansoni(27) , 27 residues obey a new consensus of 31 invariant amino acids (indicated by triangles in Fig.3). Thus, Galpha(a) harbors virtually all residues which, as deduced from their strict conservation, appear to play pivotal roles in Galpha function.

Guanine Nucleotide Binding Motifs

Intriguingly, the sequence of the A domain of the putative guanine nucleotide binding pocket of Galpha(a) is significantly different from the consensus sequence LLLLG(A/T/P)(G/S)(E/N)SGK that can be deduced from a compilation of G protein alpha subunits reported thus far. Nonetheless, it complies to the minimal consensus for this domain (GXXXXGK) as deduced from a variety of nucleotide-binding proteins(28) . The Galpha(a) C domain is also distinct, deviating from the highly conserved sequence in other Galpha proteins. First, the DVGGQR motif has been changed into DVAGQR (difference underlined), again leaving unaffected the minimal consensus sequence (DXXGQ; (28) ). Second, immediately downstream of this motif, a highly conserved glutamic acid residue has been replaced by leucine in the sequence SLRKKWIH. The unique features of the A and C domains of Galpha(a) described above add to our knowledge concerning these crucial elements of Galpha proteins (see ``Discussion''). The G and I domains of Galpha(a) are identical and similar, respectively, to analogous regions in other G protein alpha subunits.

Post-translational Modification Sites

Although not an absolute prerequisite for all Galpha proteins, N-terminal myristoylation of various G(i) class alpha subunits is supposed to enhance interaction with the Gbeta complex and the plasma membrane(29) . The Galpha(a) N terminus lacks the MGXXX(S/T) consensus required for myristoylation. In addition, it does not contain a cysteine residue that could be palmitoylated (30, 31) or modified by attachment of arachidonic acid (32) as another way of creating a hydrophobic membrane anchor. Consequently, Galpha(a) is either soluble upon dissociation of the heterotrimer or it uses other means to attach to the inner face of the plasma membrane.

The arginine residue that is conserved in all G protein alpha subunits and serves as a substrate for ADP-ribosylation by cholera toxin in Galpha(s) and Galpha(t)(1) is also present in Galpha(a). Solely on the basis of the primary structure of Galpha(a), it cannot be concluded, however, whether the protein can actually be modified by the toxin. Another bacterial toxin, pertussis toxin, modifies some G(i) class members through ADP-ribosylation of a cysteine residue four residues away from the C terminus(1) . Since Galpha(a) lacks a cysteine at this position, the G(a) protein will subserve pertussis toxin-insensitive functions.

Spatial Expression Profile of Galpha

To examine the expression of Galpha(a) within the Lymnaea central nervous system, we performed in situ hybridization experiments (Fig.4). Synthetic antisense RNA derived from the GAAL2 cDNA was used as a probe. Interestingly, the corresponding mRNA appears to be expressed in a highly cell-specific fashion in that the probe hybridizes to a limited number of neurons (less than 10%) dispersed throughout the brain (Fig.4A). For example, Galpha(a) mRNA is expressed in the so-called yellow cells, which are located, among others, in the visceral, parietal, and pleural ganglia, but also in the nerves originating from the visceral ganglia (Fig.4B; for review see (33) ). Other well studied neurons, however, like the light green cells and the caudodorsal cells in the cerebral ganglia (6) do not express Galpha(a) at a detectable level (not shown). As another example, Fig.4C shows a section of the visceral and right parietal ganglia in which cells that express Galpha(a)-encoding transcripts at a high level abut cells that do not. These data suggest that the Galpha(a) protein fulfils cell type-specific functions.


Figure 4: In situ hybridization of the Lymnaea central nervous system with a GAAL2-specific probe. Sections of the Lymnaea brain were incubated with a cRNA probe prepared from the complete GAAL2 cDNA. A, overview. C, cerebral ganglia; Pl, pleural ganglia; Pa, parietal ganglia; V, visceral ganglion; R, right; L, left. The pedal ganglia are not present in the section shown. B, detail of a visceral ganglion. Arrows indicate yellow cells within a sectioned nerve (N). C, detail of visceral (V) and right parietal (RPa) ganglia. Arrows indicate neurons that appear to highly express Galpha(a)-specific mRNA; arrow heads indicate cells that do not hybridize with the GAAL2 cRNA above background.




DISCUSSION

In this paper, we describe a novel G protein alpha subunit, Galpha(a), which is expressed differentially in the central nervous system of the pond snail, L. stagnalis. We feel confident that we have indeed cloned the alpha subunit of a heterotrimeric G protein rather than a member of the Ras family of small guanine nucleotide-binding proteins. First, its putative nucleotide-binding domains resemble those of Galpha proteins much more than those of the Ras family. This also holds true for the sequence directly following domain C, which constitutes a major conformational switch domain of G protein alpha subunits(34) . Second, its molecular mass (44.7 kDa) is within the range of Galpha proteins (39-45 kDa) but differs significantly from that of the Ras family (21 kDa). Third, Galpha(a) contains many amino acids that appear to be invariant in G protein alpha subunits. Among these is the strictly conserved arginine residue (35) (Arg-212 in Galpha(a)) that serves as a cholera toxin substrate in Galpha(s) and Galpha(t)(1) and appears to play a key role in stabilizing the transition state during GTP hydrolysis(25, 36) . Galpha(a) also contains the conserved threonine and glycine residues (Thr-217 and Gly-237), which initiate the conformational changes that occur upon guanine nucleotide exchange(34) , and the crucial glutamic acid (Glu-270) that propagates these changes to other parts in the molecule. The glutamine residue (Gln-238) that is supposed to fulfill a key role during GTP hydrolysis (25, 36) has also been preserved. Amino acid sequence comparison indicates that Galpha(a) resembles other Galpha subtypes described to date (27-33% identity) but clearly defines a distinct subclass.

Of the nucleotide-binding motifs, the A domain is involved in binding phosphate groups(24, 25) . Although the core sequence of this domain in Galpha(a) (GGPGAGK) differs considerably from known Galpha A domains, it fits the G(G/P/A)PGXGK consensus for another group of nucleotide-binding proteins, the adenylate kinases(37) . Nonetheless, Galpha subunits with A domain sequences differing from the formerly canonical GAGESGK motif exhibit deviating nucleotide affinities and kinetics of GTP hydrolysis(38, 39) . Galpha(a) may therefore exhibit a GTPase cycle with greatly distinct features.

The C domain of G protein alpha subunits coincides by and large with an important conformational switch element (switch II) (34) and is highly conserved. Surprisingly, Galpha(a) violates the consensus sequence (DVAGQR instead of DVGGQR). Such a deviation is also present in the Galpha6 and Galpha7 subunits of Dictyostelium(26) . The DVGGQR motif appears to propagate GTP-induced conformational changes to the switch II region(24, 34) . It has been suggested that the two glycines of the motif provide the necessary freedom for this action(24) . Indeed, the latter is hampered by mutation of the second glycine to alanine(40) . It is, however, highly unlikely that substitution of alanine for the first glycine has similar effects, since it has now been found to occur naturally in three independent Galpha subunits. Moreover, the strict conservation of alanine in an analogous position in Ras(-like) proteins, and the fact that mutations in this position are implicated in cellular transformation(41) , point to a functional significance of the presence of such a residue in Lymnaea Galpha(a).

Another salient deviation of Galpha(a) is found immediately downstream of the DVAGQR motif. Hitherto, the glutamic acid in the sequence SERKKWIH appeared to be strictly conserved among all Galpha subunits. On the basis of crystal structure studies, it was predicted that this residue acts as a general base during GTP hydrolysis, activating a water molecule for a nucleophilic attack on the -phosphate group(24) . However, Galpha(a) has leucine at this position, and it is very difficult to imagine that such a residue could perform a similar task. Indeed, a mutational study of the importance of the pertinent glutamic acid refutes the hypothesis(43) . Moreover, reports on the structure of GDPbulletAlF4-bound Galpha subunits (mimicking the transition state) (25, 36) support the notion that it is the conserved DVXGQR glutamine that plays an important role(35) .

Overall, the helical domain is the most heterogenous part of Galpha proteins. It is thought that this domain constitutes an independent, built-in GTPase-activating domain(35, 42) . In addition, the helical domain acts as an intrinsic guanine nucleotide dissociation inhibitor by burying the guanine nucleotide-binding pocket(24) . Its unique amino sequence in Galpha(a) suggests that there are few constraints on its (primary) structure. Such a sequence diversity might be exploited for subtype-specific interactions with other proteins.

The functions of Galpha(a) are beyond speculation as yet but are likely to involve transmembrane signaling. It will be very interesting to elucidate which receptors and effectors are coupled by this Galpha subtype. Biochemical and mutational studies of Galpha(t) and Galpha(q) have implicated the extreme C terminus, the equivalents of Galpha(a) residues 347-364, and possibly the N terminus in receptor coupling(44, 45) . The corresponding regions of Galpha(a) do not resemble their counterparts in any other Galpha protein. A similar conclusion may be drawn with respect to the effector-interaction interface. Effectors of Galpha(s) (adenylyl cyclase) and Galpha(t) (cGMP-specific phosphodiesterase) interact with discrete regions on one face of the alpha subunits(46, 47, 48) . These regions encompass their alpha2-beta4, alpha3-beta5, and alpha4-beta6 parts(24, 34) , corresponding to Galpha(a) residues 245-251, 270-297, and 330-350, respectively. Galpha(a) is significantly dissimilar in the latter two regions. Thus, although our understanding of the Galpha domains that interact with receptors and effectors is far from complete, it appears that Galpha(a) would functionally link other receptors and effectors than hitherto described Galpha subtypes. Although transmembrane signaling is an obvious possibility in terms of Galpha(a) function, it cannot be excluded that Galpha(a) is involved in other processes, like intracellular trafficking(49, 50, 51) .

Irrespective of the precise nature of its tasks, Galpha(a) appears to subserve cell-specific functions. Galpha(a)-specific mRNA is specifically expressed in a restricted set of cell types within the Lymnaea brain. Among these are the yellow cells, which are considered to be involved in osmoregulation(52) . Yet, at this stage it is hard to relate Galpha(a) expression in these cells to specific function(s). In view of the apparent cell type specificity, it will be instructing to know whether or not Galpha(a) expression is specifically restricted to the central nervous system. We also need to assess whether Galpha(a) is Lymnaea- or mollusc-specific or whether similar proteins exist in higher organisms like vertebrates. It will be very interesting to express the Galpha(a) protein and determine its biochemical properties and to perturb its function with antisense DNA or antibodies and assess its cellular role(s).

Galpha(a) is the fifth Lymnaea G protein alpha subunit identified by our group. Even the unicellular slime mold, Dictyostelium discoideum, has a Galpha family consisting of at least 8 members(26) . In Drosophila, atypical G protein alpha subunits like concertina and Galpha(f) have been discovered (22, 23) . Since the PCR might very well miss certain Galpha subtypes, especially when these subunits harbor a different sequence in the parts that were used to develop degenerate primers, we surmise that a range of Lymnaea Galpha proteins might have escaped detection. The identification of Galpha(a), a Galpha subunit with clearly distinct properties, indicates that the ever increasing array of Galpha subtypes has not yet met its limit.


FOOTNOTES

*
This study was financially supported by the Council of Geological and Biological Sciences of the Netherlands Organization for Scientific Research within the research program ``Neuropeptides and Behavior.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) Z47551[GenBank].

§
To whom correspondence should be addressed. Tel.: 31-20-4447573; Fax: 31-20-4447553; vheerik{at}chem.vu.nl.

^1
The abbreviation used is: PCR, polymerase chain reaction.

^2
E. Vreugdenhil, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Jan van Minnen for sharing expertise, critical comments, and photography of the in situ hybridizations.


REFERENCES

  1. Birnbaumer, L., Abramowitz, J., and Brown, A. M. (1990) Biochim. Biophys. Acta 1031,163-224 [Medline] [Order article via Infotrieve]
  2. Simon, M. I., Strathmann, M. P., and Gautam, N. (1991) Science 252,802-808 [Medline] [Order article via Infotrieve]
  3. Hepler, J. R., and Gilman, A. G. (1992) Trends Biochem. Sci. 17,383-387 [CrossRef][Medline] [Order article via Infotrieve]
  4. Offermans, S., and Schultz, G. (1994) Naunyn-Schmiedeberg's Arch. Pharmacol. 350,329-338
  5. Neubig, R. R. (1994) FASEB J. 8,939-946 [Abstract/Free Full Text]
  6. Geraerts, W. P. M., Smit, A. B., Li, K. W., Vreugdenhil, E., and van Heerikhuizen, H. (1991) in Current Aspects of the Neurosciences (Osborne, N. N., ed), Vol. 3, pp. 255-304, MacMillan Press, London
  7. Bulloch, A. G. M., and Syed, N. I. (1992) Trends Neurosci. 15,422-427 [CrossRef][Medline] [Order article via Infotrieve]
  8. Knol, J. C., Weidemann, W., Planta, R. J., Vreugdenhil, E., and van Heerikhuizen, H. (1992) FEBS Lett. 314,215-219 [CrossRef][Medline] [Order article via Infotrieve]
  9. Knol, J. C., Roovers, E., van Kesteren, E. R., Planta, R. J., Vreugdenhil, E., and van Heerikhuizen, H. (1994) Biochim. Biophys. Acta 1222,129-133 [Medline] [Order article via Infotrieve]
  10. Knol, J. C., Ramnatsingh, S., van Kesteren, E. R., van Minnen, J., Planta, R. J., van Heerikhuizen, H., and Vreugdenhil, E. (1995) Eur. J. Biochem 230,193-199 [Abstract]
  11. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159 [CrossRef][Medline] [Order article via Infotrieve]
  12. Strathmann, M., Wilkie, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,7407-7409 [Abstract]
  13. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  14. Gibbons, I. R., Asai, D. J., Ching, N. S., Dolecki, G. J., Mocz, G., Phillipson, C. A., Ren, H., Tang, W.-J. Y., and Gibbons, B. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8563-8567 [Abstract]
  15. Bloem, L. J., and Yu, L. (1990) Nucleic Acids Res. 18,2830 [Medline] [Order article via Infotrieve]
  16. Halliday, K. R. (1984) J. Cyclic Nucleotide Protein Phosphor. Res. 9,435-448
  17. Mount, S. R. (1982) Nucleic Acids Res. 10,459-472 [Abstract]
  18. Itoh, H., Toyama, R., Kozasa, T., Tsukamoto, T., Matsuoka, M., and Kaziro, Y. (1988) J. Biol. Chem. 263,6656-6664 [Abstract/Free Full Text]
  19. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M., and Satoh, T. (1991) Annu. Rev. Biochem. 60,349-400 [CrossRef][Medline] [Order article via Infotrieve]
  20. Lochrie, M. A., Mendel, J. E., Sternberg, P. W., and Simon, M. I. (1991) Cell Regul. 2,135-154 [Medline] [Order article via Infotrieve]
  21. Kozak, M. (1987) Nucleic Acids Res. 15,8125-8148 [Abstract]
  22. Parks, S., and Wieschaus, E. (1991) Cell 64,447-458 [Medline] [Order article via Infotrieve]
  23. Quan, F., Wolfgang, W. J., and Forte, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,4236-4240 [Abstract]
  24. Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366,654-663 [CrossRef][Medline] [Order article via Infotrieve]
  25. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265,1405-1412 [Medline] [Order article via Infotrieve]
  26. Wu, L., Gaskins, C., Zhou, K., Firtel, R. A., and Devreotes, P. N. (1994) Mol. Biol. Cell 5,691-702 [Abstract]
  27. Iltzsch, M. H., Bieber, D., Vijayasarathy, S., Webster, P., Zurita, M., Ding, J., and Mansour, T. E. (1992) J. Biol. Chem. 267,14504-14508 [Abstract/Free Full Text]
  28. Dever, T. E., Glynias, M. J., and Merrick, W. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,1814-1818 [Abstract]
  29. Spiegel, A. M., Backlund, P. S., Jr., Butrynski, J. E., Jones, T. L. Z., and Simonds, W. F. (1991) Trends Biochem. Sci. 16,338-341 [Medline] [Order article via Infotrieve]
  30. Resh, M. D. (1994) Cell 76,411-413 [Medline] [Order article via Infotrieve]
  31. Veit, M., Nürnberg, B., Spicher, K., Harteneck, C., Ponimaskin, E., Schultz, G., and Schmidt, M. F. G. (1994) FEBS Lett. 339,160-164 [CrossRef][Medline] [Order article via Infotrieve]
  32. Hallak, H., Muszbek, L., Laposata, M., Belmonte, E., Brass, L. F., and Manning, D. R. (1994) J. Biol. Chem. 269,4713-4716 [Abstract/Free Full Text]
  33. Swinddale, N. V., and Benjamin, P. R. (1976) Philos. Trans. R. Soc. Lond-Biol. Sci. 274,169-202 [Medline] [Order article via Infotrieve]
  34. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369,621-628 [CrossRef][Medline] [Order article via Infotrieve]
  35. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349,117-127 [CrossRef][Medline] [Order article via Infotrieve]
  36. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 372,276-279 [CrossRef][Medline] [Order article via Infotrieve]
  37. Saraste, M., Sibbald, P. R., and Wittinghofer, A. (1990) Trends Biochem. Sci. 15,430-434 [CrossRef][Medline] [Order article via Infotrieve]
  38. Casey, P. J., Fong, H. K. W., Simon, M. I., and Gilman, A. G. (1990) J. Biol. Chem. 265,2383-2390 [Abstract/Free Full Text]
  39. Pang, I.-H., and Sternweis, P. C. (1990) J. Biol. Chem. 265,18707-18712 [Abstract/Free Full Text]
  40. Miller, R. T., Masters, S. B., Sullivan, K. A., Beiderman, B., and Bourne, H. R. (1988) Nature 334,712-715 [CrossRef][Medline] [Order article via Infotrieve]
  41. Fasano, O., Aldrich, T., Tamanoi, F., Taparowsky, E., Furth, M., and Wigler, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,4008-4012 [Abstract]
  42. Markby, D. W., Onrust, R., and Bourne, H. R. (1993) Science 262,1895-1901 [Medline] [Order article via Infotrieve]
  43. Kleuss, C., Raw, A. S., Lee, E., Sprang, S. R., and Gilman, A. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,9828-9831 [Abstract/Free Full Text]
  44. Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B., and Hofmann, K. P. (1988) Science 241,832-835 [Medline] [Order article via Infotrieve]
  45. Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. (1993) Nature 363,274-276 [CrossRef][Medline] [Order article via Infotrieve]
  46. Berlot, C. H., and Bourne, H. R. (1992) Cell 68,911-922 [Medline] [Order article via Infotrieve]
  47. Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256,1031-1033 [Medline] [Order article via Infotrieve]
  48. Faurobert, E., Otto-Bruc, A., Chardin, P., and Chabre, M. (1993) EMBO J. 12,4191-4198 [Abstract]
  49. Barr, F. A., Leyte, A., and Huttner, W. B. (1992) Trends Cell Biol. 2,91-94 [Medline] [Order article via Infotrieve]
  50. Burgoyne, R. D. (1992) Trends Biochem. Sci. 17,87-88 [Medline] [Order article via Infotrieve]
  51. Nuoffer, C., and Balch, W. E. (1994) Annu. Rev. Biochem. 63,949-990 [CrossRef][Medline] [Order article via Infotrieve]
  52. Wendelaar Bonga, S. E. (1972) Gen. Comp. Endocrinol. , Suppl. 3, 308-316

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