- Email: [email protected]
Will the real IP4 receptor please stand up?
ROBIN F. IRVINE AND PETER J. CULLEN
INTRACELLULAR SIGNALLING
Will the real IP,, receptor
please stand up?
Several proteins have been described that bind IP, with a variety of different potencies and specificities. Amidst this confusion, is there consistent evidence for a ‘true’ IP, receptor? There has been considerable confusion and controversy over the last few years as to whether inositol 1,3,4,5tetrakisphosphate (IPq) has a second messenger role to play in the control of Caz+ entry into cells [l]. We do not intend to address the complexities of inositol phosphates and Ca2f entry here (these are nicely reviewed in 121). Instead, we shall concern ourselves mostly with the issue of whether there are specific intracellular receptors for IP4 and, if so, whether they have been identified among the many intracellular proteins that have been reported to bind IP4. Unfortunately, but perhaps not surprisingly, many intracellular proteins bind 1~4, which has only added to the confusion. We shall briefly summarize the proteins that have been reported to bind IP4 and explain why we think only one particular subset of these has the right pedigree to serve as an intracellular IP4 receptor-. IP,
of tissues (for example, Xenopz4.5ooqtes [ 1 I ) with a potency about 10-40 times less than that of IP3, and recent data from Nahorski’s laboratory [4] strongly implies that this Ca2+ -mobilizing effect of IP4 is indeed due to its direct interaction with IP3 receptors. Thus it has been argued that in tissues where some IP3 receptors are not highly discriminatory against IP~, the latter serves as an adjunct Ca2+ mobilizer [l]. We have to admit that we find this idea hard to swallow. It is entirely reasonable to suggest that using ATP to phosphorylate IP3 could be a way by which this second messenger is inactivated [ 1I ; but for cells to use up energy generating IP~ molecules that have the same effect as one tenth as many IP3 molecules would make us think that evolution has had a brainstorm! The same apparent lack of logic would apply even to a ‘receptor’ that binds IP, and IP4 with almost identical potency, such as the protein described by Khan et al. [3] in Jurkat cell plasma membranes (Fig. la). Although it is true that in some stimulated tissues IP4 levels can, over a period of time, rise higher than those of IP3, SO that one would guess IP4 could become the principal ligand bound to such a protein, this still seems an extraordinary waste of ATP. We should emphasize that all our current knowledge of inositol lipid and phosphate metabolism tells us that the only way to make IP4 is by &r&t phosphorylation of IP~, and that IP4 is catabolized at a similar rate to Ip3. Recent data from Christophe Erneux’s laboratory (C I%neux, personal communication)
receptors
Inositol 1,4,5-t&phosphate (IP3) is now well established as a second messenger, and the family of IP3 receptors is being extensively explored in many molecular and functional studies. As more information is gleaned about this family of receptors it is becoming evident that some of them bind IP4 with a higher affiity than others. For example, the microsomal IP3 receptor of thymic cells binds IP4 with an affinity only an order of magnitude less than its affinity for IP3 [3]. There are many reports that IP4 can mobilize intracellular Ca2+ in a wide range
,,, Specific binding
.~
100
(%)
1@ Concentration
1o-6
lo-”
Concentration
Concentration ./
-
INSP,
-
INSP,
-
INSP,
-
INSP 6 0
-3,
Fig. 1. Pharmacologies-of sites that are discussed&in and semi-quantitatively,
540
some IP,-binding proteins. the text. To aid simple and the cited references
@
The figure depicts visual comparison, should be consulted
Current
Biology
1993 current Biology
the displacement of IP, by IP,-, from three different sets of binding the binding displacement curves are depicted only schematically for the data and original binding curves.
1993,
Vol
3 No
8
DISPATCH
show that purilied IP3 kinase binds IP3 and IP~ at the same site with a pharmacology identical to the binding site of Jurkat cell plasma membranes, and given that IP~ kinase has been found bound to membranes [ 51 ‘we wonder whether this is what has been detected by Khan et al. _)_[31. It is now a well-established observation that IP3 receptors often co-purify with plasma membranes in subcellular fractionations (see [6,7] for references), and two recent reports assert that they are actually located within, and span, the plasma membrane [6,7]. It is often difhcult to tell whether proteins really are integral to the plasma membrane or are actually components of another membrane fraction that is associated with the plasma membrane. Both groups have acknowledged this problem, and used cell-impermeant probes in an attempt to resolve the issue. Their data suggest that there may be a very small proportion of IPQ receptors spanning the plasma membrane, though the orientation of the receptors is unknown and at these low levels it is difficult to be entirely sure that this interpretation is correct. Whether a subset of IP3 receptors are truly in the plasma membrane remains, in our opinion, open to question notwithstanding the elegant experiments of Khan et al. [6] and Fujimoto et al. [7] (and pace olfactory tissue, which seems to use all second messengers in a rather different way than most cell types). But in the context of this review what is less questionable is that any such receptors are unlikely to be involved in any effects IP4 may exert on Ca2+ entry into cells; in those experiments in which the pharmacology of these effects of IP4 has been at least semi-quantitatively explored [ 1,8], IP4 is at least an order of magnitude more potent than IP3. If we are seeking an IP4 receptor for this function, it must at the very least favour IP4 over IP3. IP6-binding
proteins
There have been several reports of inositol polyphosphate -binding proteins that do bind IP4 with a higher affinity than IP3, and the presence of these proteins has not helped to clarify matters. There is at least one protein complex that binds IP4, IPg and IPd, but interacts only very weakly with IP3; this complex was first described by Theibert et al. [9] and subsequently shown by the same group [lo] and by others [ll] to contain the clathrin assembly protein AP-2. The pharmacology of this binding site, for which IPg and IP6 have a higher ailinity than IP4 (Fig. lb), suggests that it is unlikely ever to bind IP4 in vivo (given that we currently believe IPs and IP6 have higher intracellular concentrations than IP4). The specificity of the effects of IP4 on Ca2 + -entry dS0 argues strongly that the effects cannot be mediated by this particular binding site, as IPg and IP6 are much less active in the systems studied than IP4. IP4 receptors
So what characteristics, do we require in a credible IP4 receptor? First, it should greatly favour IP4 (and the 1345 isomer at that) overany other inositol phosph.ate, such that IP4 would be its primary ligand in vivo. Second, its pharmacology should quantitatively match the effects of
IP4 on Ca2+ homeostasis [1,8]. An increasing number of tissues have been shown to exhibit a binding site with just these characteristics ([1,12] and Fig. 1~). This binding site(s) has been best characterized in the cerebellum, where its detailed pharmacology has been explored by Challiss et al. [13]; it favours the 1345 isomer of IP~ greatly over the 3456, 1456 or 1346 isomers, and binds IP3 and IPg extremely poorly. Only 13456 IPg is likely to make any contribution to displacing IP4 from these binding sites in vim although this inositol phosphate is about 50 times less potent than IP4 (Fig.&), IPg may be present at much higher levels than IP4 in?iiost tissues. However, these binding sites have been employed in ligand-displacement mass assays for IP4 in unprocessed tissue extracts in me presence of all other inositol phosphates [ 1,131, which in itself suggests there is little interference from IP,, because if that, or any other compound, were the principal ligand at these binding sites, its presence would obscure or distort the quantitative displacement of radioactive IP4 by IP4 in the cell extract. The binding assays were, however, performed under unphysiological conditions (pH 5 and 50mM inorganic phosphate), so strictly speaking the data do not clarifywhether the binding sites bind mostly IP4 in viva. We have therefore repeated the mass assays under physiological condi tions [ 141, and both the cerebellar and adrenal cortical IPq-binding sites still faithfully and quantitatively register changes in IP4, from which we infer that IP4 is indeed the primary ligand for these binding sites in intact tissues. Moreover, the pharmacology of these binding sites (Fig. lc) matches well, so far as we can quantify it, that of the effects of IP4 on Ca2+ homeostasis [ 1,8], whereas it is clearly different from any known inositol phosphatemetaboli#ng enzyme [ 121. In short, it is possible to detect in many tissues IPq-binding sites that have entirely the right qualifications to be true in vivo IP4 receptors. The molecular weight of this IP4-binding protein(s) varies with the preparation [ 11, and it is, in any case, always difficult to assign the activity to a particular band on a gel. The exception is the elegant work of Theibert et al. [ 151, in which displacement of an IP4 aflmity ligand clearly identified 182kD and 84kD proteins with the necessary pharmacological characteristics (as we are defining them here) to be true IP4 receptors. The connection between these two proteins is unclear - one may be a proteolytic cleavage product of the other [l] . If we feel that we can with some coniidence assert that there may be such things as IP4 receptors, we can begin to ask questions about their location in the cell. This has been difficult to do with currently available [3H]-IP4 probes (it is a hard task even to detect IP~ ‘receptors’ in most peripheral tissues, as they are not as abundant as IP3 receptors), but recentlywe have set up, for this purpose, a routine preparation of extremely pure [s2P] -IP4 at 4500 Ci per mmole (unpublished data). This not only makes the detection of specific binding relatively easy but, crucially, enables us fully to characterize IPq-binding sites even in isolated membrane fractions, so that we can be sure we are studying the right thing (and not one of the other IP4-binding proteins discussed above). We have applied
541
542
Current
Biology
1993,
Vol
3 No
8
this probe to highly purified platelet membrane fractions in which lPj binding had earlier been shown to fractionate predominantly with intracellular membranes [ 161. IP4 binding sites, with the right properties for being IP4 receptors (see Fig. lc), distribute entirely differently from LP~ binding sites and are clearly predominantly in the plasma membrane (PJ Cullen, RF Irvine and KS Authi, unpublished data).
Ca2+
entry
A plasma membrane location is clearly desirable if putative IP4 receptors are to mediate the effects of IP4 on CaZ+ entry into cells [ 1,81. As we have said, we cannot here go into the complexities of inositol phosphates and Ca2+ entry, but we feel we should attempt to place the putative IP4 receptors into a physiological context, particularly in relation to other mechanisms of Ca2+ entry. As Penner et al. discuss [2], there are many such mechanisms, but the principal alternative to regulation by inositol phosphates is Ca2+ pool-controlled Ca2+ entry [17]. One of us has previously tried to unite IP4 and Ca2+ poolcontrolled Ca2+ entry in a single mechanism [ 11, if only Before a Ca2”oscillationi Ca2*(@) gradually enters the.cytosol from outside the cell through lP4, receptors, and from intracellular stores n’earest these sites of Ca*+ influx:
to get around the awkward teleological question of why cells should make IP4 to control Ca2+ entry if IP3 could, by emptying intracellular Ca2+ pools, do the whole thing on its own. Various experimental data (reviewed in [ 21.) have made such an idea of a single mechanism of regulating Ca2+ entry into cells untenable, and in Figure 2 we offer one possible answer to the question of why cells might require two separate inositol phosphate-mediated mechanisms. We suggest that the two mechanisms fulfill two distinct functions. At physiological agonist doses, nonexcitable cells are in a Ca2+ ‘oscillatory’ mode [ 181. We suggest that the IP#Pq-driven Ca2+ entry is the small influx into the cytosol of Ca2+ - from both outside the cell and from peripheral intracellular stores - that drives the oscillatory mechanism [ 181. The ‘capacitative’ Ca2+ poolcontrolled mechanism, on the other hand, we suggest to be a homeostatic pool-refilling mechanism that ensures the cell is ready for the next oscillation. The mechanism of this pool-controlled entry does not in this scheme involve IP4 receptors, and how it is controlled is presently unknown. In Figure 2 we have, for lack of a
When the cytosolic CaZf concentration passes a critical level it triggers ‘dumping’. of Ca2” from all the intracellular stores into the cytosol, causing the Ca” ‘spike’.
During the recovery phase of a Ca*+’ oscillation, Ca*’ pumps reload the intracellular stores with Caz’ from the cytosol and the extracellular fluid.
,
Pool-controlled
pump
Type I IP, receptor
Fig. 2. A model summarizing our current thoughts on the role of IP, in regulating Cal+ entry into cells. We suggest that in a cell being adminstered physiological agonist doses, Ca2+ entry has two distinct functions - one is to ‘drive’ and prime the system [If% and the other is to restore the status quo rapidly after a Cal+ wave has passed through the cell, so that the system is ready for the next wave. The former, ‘priming’ function is fulfilled by the IPa/lP,-controlled mechanism governing both the release of Ca2’ from a discrete set of pools near the plasma membrane and Ca2+ entry from outside the cell; this mechanism, which may involve a distinct IPa receptor [191, continues throughout the time during which extracellular agonist receptors are activated to generate IPa and tP.+ The second, pool-refilling mechanism is controlled homeostatically by the Caz+ content of the intracellular stores, and is therefore only activated during an oscillation, when extensive mobilization of Ca2+ occurs throughout the cell.
DISPATCH
clear alternative, adapted our earlier suggestions and suggest that two proteins, one in the endoplasmic reticulum (a ryanodine receptor homologue?), which ‘senses’ the Ca2+ levels in the pools, and one in the plasma. membrane, which controls Caz+ entry into the cell, may interact dir&ly,If the capacitative Ca2+ flux is larger than the IP&P*-mediated mechanism, as it would be in most cells [IS], then it is hardly surprising that in most experiments in which IP3 is introduced into the cell by techniques such as microinjection, rather than being generated at the plasma membrane, it is impossible to detecit a significant effect of IP4 on overall Ca2+ entry. Only in cells were the capacitative entry is small, or uncoupled, will any requirement for IP4 be evident when such experimental protocols are used. There is still a great deal to learn about how inositol phosphates control Cal+ entry, and there remains much confusion in the field. But we hope that by clarifying what we define as an IF4 receptor amidst the various proteins that bind IP4 we can begin to clarify at least a part of that confusion. Achzowledgements: PJC is a Beit Memorial Fellow. Wreggett and Kalwant Authi for helpful discussions.
We thank
7.
8.
9.
10.
11.
12.
13.
Keith 14.
References 1. IRVINE RF: Is inositol that controls Ca2+ 2. 3.
4.
5.
6.
tetrakisphosphate the second messenger entry into cells? Adv Second Messenger Phosphoprotein Res 1992, 26:161-185. PENNER R, FASOLATO C, HOTH M: Calcium intlux and its control by calcium release. CUV Opin Neurobiol 1993, 3~368-375. KHAN A& STEJNER JP, SNYDER SH: Plasma membrane inositol 1,4,5trisphosphate receptor of lymphocytes: selective enrichment in sialic acid and unique binding specificity. Proc Nat1 Acud Sci USA 1992, 89:28492853. WILCOX R& CHALLIS RAJ, BAUDIN G, VASELIA V, POTTER BVL, NAHORXI SR: Stereoselectivity of Ins(1,3,4,5)P* recognition sites: implications for the mechanism of Ins(1,3,4,5)Phinduced CaZ+ mobilization. Biocbem J 1993, 294:191-194. MORRIS AJ, DO~NES CP, HARDEN TR, MICHELL RH: Turkey erythrocytes possess a membrane-associated inositol 1,4,5t&phosphate 3-kinase that is activated by Caz+ in the presence of calmodulin. Biocbem J 1987, 248:48ti93. KHAN fi STEINER JP, KLEIN MG, SCHNEIDER MF, SNYDER SH: IP3 receptor: localization to plasma membrane of T cells and cocapping with the T-cell receptor. Science 1992, 257:815-818.
15.
16.
17. 18. 19.
FUJIMOTO T, NAKADA S, MryAwAKI A, MIKOSHIBA K, OGAWA K: Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Bioll992, 119~1507-1513. LUCKHOFF 4 CLAPHAM D: Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca2+ -permeable channel. Nature 1992, 355:35&358. ~IEIBERT AB, ESTEVEZ VA, FERRLS CD, DANOFF SK, BARROW RK, PRESTW~CH GD, SNYDER SH: Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterisation from rat brains. Proc Nut1 Acud Sci USA 1991, 85~3165-3169. V~GLMAIER SM, KEEN JH, MURPHY JE, FERRIS CD, PRESTWICK GD, ShWsR SH, THEIBERT AB: Inositol hexakisphosphate receptor identified as the clatbrin assembly protein AP-2. Biocbem Bioplys Res Commun 1992, 187:158-163. TIMERAN AF’, MAyRLEmR MM, LUKAS TJ, CHADwICK CC, SAlTO A, WA’ITERSON DM, SCHINDLER M, FLEISCHER S: Inositol polyphosphate receptor and clathrin assembly protein AP-2 are related proteins that form potassium-selective ion channels in plasma ligand bilayers. Proc NatE Acad Sci USA 1992, 89:89768980. BRADFORD PG, IRVINE RF: Specific binclmg sites for [3H] inositol (1,3,4,5) tetrakisphosphate on membranes of HI-60 cells. Bzochem Biopbys Res Commun 1987, 149:680-685. CHALUSS RAJ, WILLOCKS AC, MULLOY B, POTTER BVL, NAHORXI SR: Characterization of inositol 1,4,5Msphosphate and inositol I,3,4,5-tetrakisphosphate-binding sites in rat cerebellum. Biochem J 1991, 274:861+367. CULLEN PJ, IRW RF: Inositol 1,3,4,5-tetrakisphosphate binding sites in neuronal and non-neuronal tissues. Properties, comparisons and potential physiological significance. Biocbem J 1992, 288:149154. THEIBERT AB, ESTEVEZ VA, MOUREY RJ, MARECEK JF, BARROW RK, PRESTWICH GD, SNYDER SH: Photoaffinity Iabelling and characterisation of isolated inositol 1,3,4,5-tetraldsphosphate and inositol hexakisphosphate-binding proteins. J Biol Gem 1992, 267:9071-9079. ALITHI KS: Localisation of the [32P]-IP3 bmding site on human platelet intracellular membranes isolated by high-voltage freeflow electrophoresis. FEBS Letters 1992, 298:173-176. PUT~XEY JW: Capacitative calcium entry revisited. Cell Calcium 1990, 11:611624. BERRIDGE MJ: Inositol trisphosphate and calcium SignaIIing. Nature 1993, 361:315-325. SUDHOF TC, NEWTON GL, ARCHER BT III, USDHKARYOV Y4 ~!&GNERY GA: Structure of a novel Ins P3 receptor. EM30 J 1991, 10:3199-3206.
Robin F. Irvine and Peter J. Cullen, AFRC Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
543
INTRACELLULAR SIGNALLING
Will the real IP,, receptor
please stand up?
Several proteins have been described that bind IP, with a variety of different potencies and specificities. Amidst this confusion, is there consistent evidence for a ‘true’ IP, receptor? There has been considerable confusion and controversy over the last few years as to whether inositol 1,3,4,5tetrakisphosphate (IPq) has a second messenger role to play in the control of Caz+ entry into cells [l]. We do not intend to address the complexities of inositol phosphates and Ca2f entry here (these are nicely reviewed in 121). Instead, we shall concern ourselves mostly with the issue of whether there are specific intracellular receptors for IP4 and, if so, whether they have been identified among the many intracellular proteins that have been reported to bind IP4. Unfortunately, but perhaps not surprisingly, many intracellular proteins bind 1~4, which has only added to the confusion. We shall briefly summarize the proteins that have been reported to bind IP4 and explain why we think only one particular subset of these has the right pedigree to serve as an intracellular IP4 receptor-. IP,
of tissues (for example, Xenopz4.5ooqtes [ 1 I ) with a potency about 10-40 times less than that of IP3, and recent data from Nahorski’s laboratory [4] strongly implies that this Ca2+ -mobilizing effect of IP4 is indeed due to its direct interaction with IP3 receptors. Thus it has been argued that in tissues where some IP3 receptors are not highly discriminatory against IP~, the latter serves as an adjunct Ca2+ mobilizer [l]. We have to admit that we find this idea hard to swallow. It is entirely reasonable to suggest that using ATP to phosphorylate IP3 could be a way by which this second messenger is inactivated [ 1I ; but for cells to use up energy generating IP~ molecules that have the same effect as one tenth as many IP3 molecules would make us think that evolution has had a brainstorm! The same apparent lack of logic would apply even to a ‘receptor’ that binds IP, and IP4 with almost identical potency, such as the protein described by Khan et al. [3] in Jurkat cell plasma membranes (Fig. la). Although it is true that in some stimulated tissues IP4 levels can, over a period of time, rise higher than those of IP3, SO that one would guess IP4 could become the principal ligand bound to such a protein, this still seems an extraordinary waste of ATP. We should emphasize that all our current knowledge of inositol lipid and phosphate metabolism tells us that the only way to make IP4 is by &r&t phosphorylation of IP~, and that IP4 is catabolized at a similar rate to Ip3. Recent data from Christophe Erneux’s laboratory (C I%neux, personal communication)
receptors
Inositol 1,4,5-t&phosphate (IP3) is now well established as a second messenger, and the family of IP3 receptors is being extensively explored in many molecular and functional studies. As more information is gleaned about this family of receptors it is becoming evident that some of them bind IP4 with a higher affiity than others. For example, the microsomal IP3 receptor of thymic cells binds IP4 with an affinity only an order of magnitude less than its affinity for IP3 [3]. There are many reports that IP4 can mobilize intracellular Ca2+ in a wide range
,,, Specific binding
.~
100
(%)
1@ Concentration
1o-6
lo-”
Concentration
Concentration ./
-
INSP,
-
INSP,
-
INSP,
-
INSP 6 0
-3,
Fig. 1. Pharmacologies-of sites that are discussed&in and semi-quantitatively,
540
some IP,-binding proteins. the text. To aid simple and the cited references
@
The figure depicts visual comparison, should be consulted
Current
Biology
1993 current Biology
the displacement of IP, by IP,-, from three different sets of binding the binding displacement curves are depicted only schematically for the data and original binding curves.
1993,
Vol
3 No
8
DISPATCH
show that purilied IP3 kinase binds IP3 and IP~ at the same site with a pharmacology identical to the binding site of Jurkat cell plasma membranes, and given that IP~ kinase has been found bound to membranes [ 51 ‘we wonder whether this is what has been detected by Khan et al. _)_[31. It is now a well-established observation that IP3 receptors often co-purify with plasma membranes in subcellular fractionations (see [6,7] for references), and two recent reports assert that they are actually located within, and span, the plasma membrane [6,7]. It is often difhcult to tell whether proteins really are integral to the plasma membrane or are actually components of another membrane fraction that is associated with the plasma membrane. Both groups have acknowledged this problem, and used cell-impermeant probes in an attempt to resolve the issue. Their data suggest that there may be a very small proportion of IPQ receptors spanning the plasma membrane, though the orientation of the receptors is unknown and at these low levels it is difficult to be entirely sure that this interpretation is correct. Whether a subset of IP3 receptors are truly in the plasma membrane remains, in our opinion, open to question notwithstanding the elegant experiments of Khan et al. [6] and Fujimoto et al. [7] (and pace olfactory tissue, which seems to use all second messengers in a rather different way than most cell types). But in the context of this review what is less questionable is that any such receptors are unlikely to be involved in any effects IP4 may exert on Ca2+ entry into cells; in those experiments in which the pharmacology of these effects of IP4 has been at least semi-quantitatively explored [ 1,8], IP4 is at least an order of magnitude more potent than IP3. If we are seeking an IP4 receptor for this function, it must at the very least favour IP4 over IP3. IP6-binding
proteins
There have been several reports of inositol polyphosphate -binding proteins that do bind IP4 with a higher affinity than IP3, and the presence of these proteins has not helped to clarify matters. There is at least one protein complex that binds IP4, IPg and IPd, but interacts only very weakly with IP3; this complex was first described by Theibert et al. [9] and subsequently shown by the same group [lo] and by others [ll] to contain the clathrin assembly protein AP-2. The pharmacology of this binding site, for which IPg and IP6 have a higher ailinity than IP4 (Fig. lb), suggests that it is unlikely ever to bind IP4 in vivo (given that we currently believe IPs and IP6 have higher intracellular concentrations than IP4). The specificity of the effects of IP4 on Ca2 + -entry dS0 argues strongly that the effects cannot be mediated by this particular binding site, as IPg and IP6 are much less active in the systems studied than IP4. IP4 receptors
So what characteristics, do we require in a credible IP4 receptor? First, it should greatly favour IP4 (and the 1345 isomer at that) overany other inositol phosph.ate, such that IP4 would be its primary ligand in vivo. Second, its pharmacology should quantitatively match the effects of
IP4 on Ca2+ homeostasis [1,8]. An increasing number of tissues have been shown to exhibit a binding site with just these characteristics ([1,12] and Fig. 1~). This binding site(s) has been best characterized in the cerebellum, where its detailed pharmacology has been explored by Challiss et al. [13]; it favours the 1345 isomer of IP~ greatly over the 3456, 1456 or 1346 isomers, and binds IP3 and IPg extremely poorly. Only 13456 IPg is likely to make any contribution to displacing IP4 from these binding sites in vim although this inositol phosphate is about 50 times less potent than IP4 (Fig.&), IPg may be present at much higher levels than IP4 in?iiost tissues. However, these binding sites have been employed in ligand-displacement mass assays for IP4 in unprocessed tissue extracts in me presence of all other inositol phosphates [ 1,131, which in itself suggests there is little interference from IP,, because if that, or any other compound, were the principal ligand at these binding sites, its presence would obscure or distort the quantitative displacement of radioactive IP4 by IP4 in the cell extract. The binding assays were, however, performed under unphysiological conditions (pH 5 and 50mM inorganic phosphate), so strictly speaking the data do not clarifywhether the binding sites bind mostly IP4 in viva. We have therefore repeated the mass assays under physiological condi tions [ 141, and both the cerebellar and adrenal cortical IPq-binding sites still faithfully and quantitatively register changes in IP4, from which we infer that IP4 is indeed the primary ligand for these binding sites in intact tissues. Moreover, the pharmacology of these binding sites (Fig. lc) matches well, so far as we can quantify it, that of the effects of IP4 on Ca2+ homeostasis [ 1,8], whereas it is clearly different from any known inositol phosphatemetaboli#ng enzyme [ 121. In short, it is possible to detect in many tissues IPq-binding sites that have entirely the right qualifications to be true in vivo IP4 receptors. The molecular weight of this IP4-binding protein(s) varies with the preparation [ 11, and it is, in any case, always difficult to assign the activity to a particular band on a gel. The exception is the elegant work of Theibert et al. [ 151, in which displacement of an IP4 aflmity ligand clearly identified 182kD and 84kD proteins with the necessary pharmacological characteristics (as we are defining them here) to be true IP4 receptors. The connection between these two proteins is unclear - one may be a proteolytic cleavage product of the other [l] . If we feel that we can with some coniidence assert that there may be such things as IP4 receptors, we can begin to ask questions about their location in the cell. This has been difficult to do with currently available [3H]-IP4 probes (it is a hard task even to detect IP~ ‘receptors’ in most peripheral tissues, as they are not as abundant as IP3 receptors), but recentlywe have set up, for this purpose, a routine preparation of extremely pure [s2P] -IP4 at 4500 Ci per mmole (unpublished data). This not only makes the detection of specific binding relatively easy but, crucially, enables us fully to characterize IPq-binding sites even in isolated membrane fractions, so that we can be sure we are studying the right thing (and not one of the other IP4-binding proteins discussed above). We have applied
541
542
Current
Biology
1993,
Vol
3 No
8
this probe to highly purified platelet membrane fractions in which lPj binding had earlier been shown to fractionate predominantly with intracellular membranes [ 161. IP4 binding sites, with the right properties for being IP4 receptors (see Fig. lc), distribute entirely differently from LP~ binding sites and are clearly predominantly in the plasma membrane (PJ Cullen, RF Irvine and KS Authi, unpublished data).
Ca2+
entry
A plasma membrane location is clearly desirable if putative IP4 receptors are to mediate the effects of IP4 on CaZ+ entry into cells [ 1,81. As we have said, we cannot here go into the complexities of inositol phosphates and Ca2+ entry, but we feel we should attempt to place the putative IP4 receptors into a physiological context, particularly in relation to other mechanisms of Ca2+ entry. As Penner et al. discuss [2], there are many such mechanisms, but the principal alternative to regulation by inositol phosphates is Ca2+ pool-controlled Ca2+ entry [17]. One of us has previously tried to unite IP4 and Ca2+ poolcontrolled Ca2+ entry in a single mechanism [ 11, if only Before a Ca2”oscillationi Ca2*(@) gradually enters the.cytosol from outside the cell through lP4, receptors, and from intracellular stores n’earest these sites of Ca*+ influx:
to get around the awkward teleological question of why cells should make IP4 to control Ca2+ entry if IP3 could, by emptying intracellular Ca2+ pools, do the whole thing on its own. Various experimental data (reviewed in [ 21.) have made such an idea of a single mechanism of regulating Ca2+ entry into cells untenable, and in Figure 2 we offer one possible answer to the question of why cells might require two separate inositol phosphate-mediated mechanisms. We suggest that the two mechanisms fulfill two distinct functions. At physiological agonist doses, nonexcitable cells are in a Ca2+ ‘oscillatory’ mode [ 181. We suggest that the IP#Pq-driven Ca2+ entry is the small influx into the cytosol of Ca2+ - from both outside the cell and from peripheral intracellular stores - that drives the oscillatory mechanism [ 181. The ‘capacitative’ Ca2+ poolcontrolled mechanism, on the other hand, we suggest to be a homeostatic pool-refilling mechanism that ensures the cell is ready for the next oscillation. The mechanism of this pool-controlled entry does not in this scheme involve IP4 receptors, and how it is controlled is presently unknown. In Figure 2 we have, for lack of a
When the cytosolic CaZf concentration passes a critical level it triggers ‘dumping’. of Ca2” from all the intracellular stores into the cytosol, causing the Ca” ‘spike’.
During the recovery phase of a Ca*+’ oscillation, Ca*’ pumps reload the intracellular stores with Caz’ from the cytosol and the extracellular fluid.
,
Pool-controlled
pump
Type I IP, receptor
Fig. 2. A model summarizing our current thoughts on the role of IP, in regulating Cal+ entry into cells. We suggest that in a cell being adminstered physiological agonist doses, Ca2+ entry has two distinct functions - one is to ‘drive’ and prime the system [If% and the other is to restore the status quo rapidly after a Cal+ wave has passed through the cell, so that the system is ready for the next wave. The former, ‘priming’ function is fulfilled by the IPa/lP,-controlled mechanism governing both the release of Ca2’ from a discrete set of pools near the plasma membrane and Ca2+ entry from outside the cell; this mechanism, which may involve a distinct IPa receptor [191, continues throughout the time during which extracellular agonist receptors are activated to generate IPa and tP.+ The second, pool-refilling mechanism is controlled homeostatically by the Caz+ content of the intracellular stores, and is therefore only activated during an oscillation, when extensive mobilization of Ca2+ occurs throughout the cell.
DISPATCH
clear alternative, adapted our earlier suggestions and suggest that two proteins, one in the endoplasmic reticulum (a ryanodine receptor homologue?), which ‘senses’ the Ca2+ levels in the pools, and one in the plasma. membrane, which controls Caz+ entry into the cell, may interact dir&ly,If the capacitative Ca2+ flux is larger than the IP&P*-mediated mechanism, as it would be in most cells [IS], then it is hardly surprising that in most experiments in which IP3 is introduced into the cell by techniques such as microinjection, rather than being generated at the plasma membrane, it is impossible to detecit a significant effect of IP4 on overall Ca2+ entry. Only in cells were the capacitative entry is small, or uncoupled, will any requirement for IP4 be evident when such experimental protocols are used. There is still a great deal to learn about how inositol phosphates control Cal+ entry, and there remains much confusion in the field. But we hope that by clarifying what we define as an IF4 receptor amidst the various proteins that bind IP4 we can begin to clarify at least a part of that confusion. Achzowledgements: PJC is a Beit Memorial Fellow. Wreggett and Kalwant Authi for helpful discussions.
We thank
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Robin F. Irvine and Peter J. Cullen, AFRC Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
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