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Biomedical subjects

R K Andrews

Publications and source records attributed to R K Andrews.

At least 19 recordsLinked to original sources
PubMed2003

Glycoprotein Ib-IX-V.

Glycoprotein (GP) Ib-IX-V is a remarkable platelet adhesion receptor of the leucine-rich repeat family. It has evolved to fulfil its major function of initiating platelet aggregation (thrombus formation) at high-shear stress in flowing blood. In addition to binding von Willebrand factor (vWF) in subendothelial matrix or plasma to trigger platelet aggregation, GPIb-IX-V also binds counter-receptors, alphaMbeta2 (Mac-1) on neutrophils or P-selectin on activated platelets or endothelial cells. GPIb-IX-V ligands also include alpha-thrombin, clotting factors XI/XIIa, and high-molecular-weight kininogen. Interactions involving GPIb-IX-V are therefore central to vascular processes of thrombosis and inflammation, and the receptor is under intense scrutiny as a potential therapeutic target.

Animals↗
PubMed2001

Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G.

In the inflammatory response, leukocyte rolling before adhesion and transmigration through the blood vessel wall is mediated by specific cell surface adhesion receptors. Neutrophil rolling involves the interaction of P-selectin expressed on activated endothelium and its counter-receptor on neutrophils, P-selectin glycoprotein ligand-1 (PSGL-1). Here, it is reported that P-selectin binding to neutrophils is lost under conditions that cause the release of proteinases from neutrophil primary granules. Treatment of neutrophils with the purified neutrophil granule proteinases, cathepsin G and elastase, rapidly abolished their capacity to bind P-selectin. This inactivation corresponded to loss of the N-terminal domain of PSGL-1, as assessed by Western blot analysis. A loss of intact PSGL-1 protein from the surfaces of neutrophils after the induction of degranulation was also detected by Western blot analysis. Cathepsin G initially cleaved near the PSGL-1 N-terminus, whereas neutrophil elastase predominantly cleaved at a more C-terminal site within the protein mucin core. Consistent with this, cathepsin G cleaved a synthetic peptide based on the PSGL-1 N-terminus between Tyr-7/Leu-8. Under conditions producing neutrophil degranulation in incubations containing mixtures of platelets and neutrophils, the loss of PSGL-1, but not P-selectin, from platelet-neutrophil lysates was detected. Cathepsin G- or neutrophil elastase-mediated PSGL-1 proteolysis may constitute a potential autocrine mechanism for down-regulation of neutrophil adhesion to P-selectin.

Amino Acid Sequence↗
PubMed2001

Interaction of calmodulin with the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex.

Engagement of platelet membrane glycoprotein (GP) Ib-IX-V by von Willebrand factor triggers Ca(++)-dependent activation of alphaIIbbeta3, resulting in (patho)physiological thrombus formation. It is demonstrated here that the cytoplasmic domain of GPIb-IX-V associates with cytosolic calmodulin. First, an anti-GPIbalpha antibody coimmunoprecipitated GPIb-IX and calmodulin from platelet lysates. Following platelet stimulation, calmodulin dissociated from GPIb-IX and, like the GPIb-IX-associated proteins 14-3-3zeta and p85, redistributed to the activated cytoskeleton. Second, a synthetic peptide based on the cytoplasmic sequence of GPIbbeta, R149-L167 (single-letter amino acid codes), affinity-isolated calmodulin from platelet cytosol in the presence of Ca(++) as confirmed by comigration with bovine calmodulin on sodium dodecyl sulfate-polyacrylamide gels, by sequence analysis, and by immunoreactivity with the use of an anticalmodulin antibody. The membrane-proximal GPIbbeta sequence was analogous to a previously reported calmodulin-binding sequence in the leukocyte adhesion receptor, L-selectin. In addition, the cytoplasmic sequence of GPV, K529-G544, was analogous to a calmodulin-binding IQ motif within the alpha1c subunit of L-type Ca(++) channels. Calmodulin coimmunoprecipitated with GPV from resting platelet lysates, but was dissociated in stimulated platelets. A GPV-related synthetic peptide also bound calmodulin and induced a Ca(++)-dependent shift on nondenaturing gels. Together, these results suggest separate regions of GPIb-IX-V can directly bind calmodulin, and this novel interaction potentially regulates aspects of GPIb-IX-V-dependent platelet activation. (Blood. 2001;98:681-687)

Amino Acid Motifs↗
PubMed2001

The snake venom toxin alboaggregin-A activates glycoprotein VI.

The glycoprotein (GP)-Ib-IX-V receptor complex has recently been reported to signal through a pathway similar to that used by the collagen receptor GPVI, with a critical role described for the Fc receptor gamma-chain. The evidence for this was based in part on studies with the GPIbalpha-selective snake venom toxin, alboaggregin-A. In the present study, it is reported that alboaggregin-A has activity at the collagen receptor GPVI in addition to GPIbalpha, and evidence is provided that this contributes to protein tyrosine phosphorylation, shape change, and GPIIb-IIIa-dependent aggregation. This may explain why responses to alboaggregin-A are distinct from those to von Willebrand factor-ristocetin. (Blood. 2001;97:3989-3991)

Blood Platelets↗
PubMed2001

A novel viper venom metalloproteinase, alborhagin, is an agonist at the platelet collagen receptor GPVI.

The interaction of platelet membrane glycoprotein VI (GPVI) with collagen can initiate (patho)physiological thrombus formation. The viper venom C-type lectin family proteins convulxin and alboaggregin-A activate platelets by interacting with GPVI. In this study, we isolated from white-lipped tree viper (Trimeresurus albolabris) venom, alborhagin, which is functionally related to convulxin because it activates platelets but is structurally different and related to venom metalloproteinases. Alborhagin-induced platelet aggregation (EC50, <7.5 microg/ml) was inhibitable by an anti-alphaIIbbeta3 antibody, CRC64, and the Src family kinase inhibitor PP1, suggesting that alborhagin activates platelets, leading to alphaIIbbeta3-dependent aggregation. Additional evidence suggested that, like convulxin, alborhagin activated platelets by a mechanism involving GPVI. First, alborhagin- and convulxin-treated platelets showed a similar tyrosine phosphorylation pattern, including a similar level of phospholipase Cgamma2 phosphorylation. Second, alborhagin induced GPVI-dependent responses in GPVI-transfected K562 and Jurkat cells. Third, alborhagin-dependent aggregation of mouse platelets was inhibited by the anti-GPVI monoclonal antibody JAQ1. Alborhagin had minimal effect on convulxin binding to GPVI-expressing cells, indicating that these venom proteins may recognize distinct binding sites. Characterization of alborhagin as a GPVI agonist that is structurally distinct from convulxin demonstrates the versatility of snake venom toxins and provides a novel probe for GPVI-dependent platelet activation.

Amino Acid Sequence↗
PubMed2001

Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with shear-dependent interactions.

Under conditions of high shear stress, both hemostasis and thrombosis are initiated by the interaction of the platelet membrane glycoprotein (GP) Ib-IX-V complex with its adhesive ligand, von Willebrand factor (vWF), in the subendothelial matrix or plasma. This interaction involves the A1 domain of vWF and the N-terminal extracellular region of GP Ibalpha (His-1-Glu-282), and it can also be induced under static conditions by the modulators ristocetin and botrocetin. In this study, a panel of anti-vWF and anti-GP Ibalpha antibodies-previously characterized for their effects on ristocetin- and botrocetin-dependent vWF-GP Ib-IX-V interactions-was analyzed for their capacity to inhibit either the adhesion of Chinese hamster ovary cells expressing recombinant GP Ibalpha to surface-associated vWF under hydrodynamic flow or shear-stress-induced platelet aggregation. The combined results suggest that the shear-dependent interactions between vWF and GP Ibalpha closely correlate with ristocetin- rather than botrocetin-dependent binding under static conditions and that certain anti-vWF monoclonal antibodies are able to selectively inhibit shear-dependent platelet aggregation.

Antibodies, Monoclonal↗
PubMed2001

Platelet adhesion receptors and (patho)physiological thrombus formation.

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.

Blood Platelets↗
PubMed2001

The vascular biology of the glycoprotein Ib-IX-V complex.

It has long been recognized that binding of von Willebrand factor (vWf) by the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates the cascade of events leading to thrombosis and haemostasis. In the last several years, however, it has become apparent that the GP Ib-IX-V complex plays a much broader role in vascular biology, a recognition in part due to the identification of novel adhesive ligands for GP Ib-IX-V such as P-selectin and Mac-1. Binding of vWf by the GP Ib-IX-V complex is also important in formation of the developing thrombus. Platelets can utilize the GP Ib-IX-V complex and PSGL-1 to roll on activated endothelium via surface-expressed endothelial P-selectin. The GP Ib-IX-V complex can bind the I domain of Mac-1, a binding interaction potentially relevant to transmigration of macrophages through mural thrombus, a process required for vessel remodeling post angioplasty. This review will focus on recent advances in our understanding of the structure and function of this important platelet receptor, with particular emphasis on insights made within the last two years.

Endothelium, Vascular↗
PubMed2000

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets.

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.

Antibodies, Monoclonal↗
PubMed2000

Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions.

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.

Amino Acid Sequence↗
PubMed2000

Requirement of leucine-rich repeats of glycoprotein (GP) Ibalpha for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib-IX-V complex.

The platelet glycoprotein (GP) Ib-IX-V complex mediates adhesion to von Willebrand factor (vWf) in (patho)physiologic thrombus formation. The vWf-binding site on GP Ib-IX-V is within the N-terminal 282 residues of GP Ibalpha, which consist of an N-terminal flanking sequence (His-1-Ile-35), 7 leucine-rich repeats (Leu-36-Ala-200), a C-terminal flank (Phe-201-Gly-268), and a sulfated tyrosine sequence (Asp-269-Glu-282). We have used mammalian cell expression of canine-human chimeras of GP Ibalpha, corresponding to precise structural boundaries, to demonstrate the first specific requirement for individual leucine-rich repeats for binding of vWf either induced by a modulator, ristocetin, or under hydrodynamic flow. Implicit in this approach was that the GP Ibalpha chimeras retained a functional conformation, a supposition confirmed by analyzing restoration of function to reversed human-canine chimeras and demonstrating that all chimeras bound vWf activated by botrocetin, a modulator that is indiscriminate between species. Leucine-rich repeats 2, 3, and 4 of GP Ibalpha were identified as being critical for vWf adhesion to GP Ib-IX-V.

Animals↗
PubMed2000

Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation.

Binding of the adhesive glycoprotein, von Willebrand factor (vWf), to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates platelet adhesion and aggregation at high shear stress in hemostasis and thrombosis. In this study, the GP Ib-IX-V binding site within the vWf A1 domain was analyzed using a panel of murine monoclonal antibodies raised against a 39/34-kd vWf fragment (Leu-480/Val-481-Gly-718) encompassing the A1 domain. One antibody, 6G1, strongly inhibited ristocetin-dependent vWf binding to platelets, but had no effect on botrocetin- or jaracetin-dependent binding, or asialo-vWf-dependent platelet aggregation. The 6G1 epitope was mapped to Glu-700-Asp-709, confirming the importance of this region for modulation of vWf by ristocetin. Like ristocetin, 6G1 activated the vWf A1 domain, because it enhanced binding of the 39/34-kd fragment to platelets. In contrast, 5D2 and CR1 completely inhibited asialo-vWf-induced platelet aggregation and ristocetin-induced vWf binding to GP Ib-IX-V. However, only 5D2 blocked botrocetin- and jaracetin-induced vWf binding to platelets and binding of vWf to botrocetin- and jaracetin-coated beads. Epitopes for 5D2 and CR1 were conformationally dependent, but not congruent. Other antibodies mapped to epitopes within the A1 domain (CR2 and CR15, Leu-494-Leu-512; CR2, Phe-536-Ala-554; CR3, Arg-578-Glu-596; CR11 and CR15, Ala-564-Ser-582) were not functional, identifying regions of the vWf A1 domain not directly involved in vWf-GP Ib-IX-V interaction. The combined results provide evidence that the proline-rich sequence Glu-700-Asp-709 constitutes a regulatory site for ristocetin, and that ristocetin and botrocetin induce, at least in part, separate receptor-recognition sites on vWf. (Blood. 2000;95:164-172)

Amino Acid Sequence↗
PubMed2000

Snake venom modulators of platelet adhesion receptors and their ligands.

In thrombosis, platelet aggregation is initiated by a specific membrane glycoprotein (GP) Ib-IX-V complex binding to its adhesive ligand, von Willebrand factor, in the matrix of ruptured atherosclerotic plaques or in plasma exposed to high hydrodynamic shear stress. This process closely resembles normal haemostasis at high shear, where GP Ib-IX-V-dependent platelet adhesion to von Willebrand factor in the injured blood vessel wall initiates platelet activation and integrin alphaIIb beta3 (GP IIb-IIIa)-dependent platelet aggregation. At low shear, other receptors such as those that bind collagen, the integrin alpha2beta1 (GP Ia-IIa) or GP VI, mediate platelet adhesion. Recently, snake venom proteins have been identified that selectively modulate platelet function, either promoting or inhibiting platelet aggregation by targeting GP Ib-IX-V, alpha2beta1, GP VI, alphaIIb beta3, or their respective ligands. Interestingly, these venom proteins typically belong to one of two major protein families, the C-type lectin family or the metalloproteinase-disintegrins. This review focuses on recent insights into structure-activity relationships of snake venom proteins that regulate platelet function, and the ways in which these novel probes have contributed in unexpected ways to our understanding of the molecular mechanisms underlying thrombosis.

Amino Acid Sequence↗
PubMed1999

Identification of a 14-3-3 binding sequence in the common beta chain of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors that is serine-phosphorylated by GM-CSF.

The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.

14-3-3 Proteins↗
PubMed1998

Binding of purified 14-3-3 zeta signaling protein to discrete amino acid sequences within the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX-V complex.

The glycoprotein (GP) Ib-IX-V complex constitutively expressed on the platelet plasma membrane mediates initial adhesion of circulating platelets to vessel wall matrix at high shear, and shear-induced platelet aggregation. In both cases, this involves binding of GP Ib-IX-V to the adhesive glycoprotein, von Willebrand Factor (vWF). vWF binding to GP Ib-IX-V rapidly induces platelet activation, leading to cytoskeletal rearrangement, shape change, and secretion that enables alphaIIbbeta3 integrin (GP IIb-IIIa)-dependent platelet aggregation. All these events are critical in (patho)physiological thrombus formation. The recent discovery that the signaling protein, 14-3-3 zeta, copurifies with the GP Ib-IX complex (minus GP V) [Du, X., Harris, S. J., Tetaz, T. J., Ginsberg, M. H., & Berndt, M. C. (1994) J. Biol. Chem. 269, 18287-18290] indicated a potential mechanism for vWF-dependent signaling. The aim of the present study was to identify discrete amino acid sequences that bind 14-3-3 zeta within the cytoplasmic domain of the receptor. As an initial screening assay, overlapping synthetic peptides based on the cytoplasmic domains of GP Ibalpha (100 residues), GP Ibbeta (34 residues), GP IX (5 residues), and GP V (16 residues) were immobilized and assessed for the ability to bind purified 14-3-3 zeta. The C-terminal sequence GHSL of GP Ibalpha was identified as one 14-3-3 zeta interactive sequence, consistent with previous results [Du, X., Fox, J. E., & Pei, S. (1996) J. Biol. Chem. 271, 7362-7367]. Binding of 125I-labeled 14-3-3 zeta to GHSL-containing peptides was inhibitable by unlabeled 14-3-3 zeta and by anti-14-3-3 zeta IgG. Ala-walking through the GHSL sequence suggested all residues were necessary for optimal binding. In addition, 14-3-3 zeta bound with lower affinity to a peptide based on the central region of the GP Ibalpha cytoplasmic domain (Arg-557-Gly-575), whereas peptide sequences within the cytoplasmic domains of GP Ibbeta (Arg-160-Arg-175) and GP V (Lys-529-Gly-544) bound 14-3-3 zeta with comparable affinity to the GHSL-containing peptide. Soluble GHSL-containing peptides, GP Ibbeta- and GP V-based peptides semidissociated 14-3-3 zeta from GP Ib-IX-V or GP Ib-IX in platelet extracts as analyzed by immunoprecipitation, suggesting these sequences, at least partially, mediate the GP Ib-IX-V-14-3-3 zeta interaction in cells. Further, phosphorylation of the GP Ibbeta peptide at a site corresponding to a protein kinase A phosphorylation site (Ser-166) enhanced the affinity of 14-3-3 zeta binding by approximately 8-fold, suggesting phosphorylation as a potential mechanism for regulating 14-3-3 zeta association with the GP Ib-IX-V complex.

14-3-3 Proteins↗
PubMed1998

Conversion of forskolin-insensitive to forskolin-sensitive (mouse-type IX) adenylyl cyclase.

Forskolin potently activates all cloned mammalian adenylyl cyclases except type IX by interacting with two homologous cytoplasmic domains (C1 and C2) that form the catalytic core. A mutational analysis of the IIC2 protein (C2 domain from type II adenylyl cyclase) and forskolin analogs suggests that Ser942 interacts with the 7-acetyl group of forskolin. The C1/C2 complex has only one forskolin, one ATP, and one binding site for the alpha subunit of the G protein that stimulates adenylyl cyclase (Gsalpha) and its structure may be modeled using the three-dimensional structure of (IIC2/forskolin)2. The Ser942 mutation defines which forskolin in the (IIC2/forskolin)2 structure exists in C1/C2 complex. Thus, the forskolin-binding site is close to the Gsalpha-binding site but distal (15-20A) from the catalytic site. Mutation from Leu912 of IIC2 protein to tyrosine or alanine severely reduces Gsalpha activation and completely prevents forskolin activation. The corresponding residue of Leu912 is Tyr1082 at type IX isoform of adenylyl cyclase. Similar to recombinant type IX enzyme, soluble adenylyl cyclase derived from mouse-type IX adenylyl cyclase is sensitive to Gsalpha activation but not to forskolin. Changing Tyr1082 to leucine makes soluble type IX adenylyl cyclase forskolin-responsive.

Adenylyl Cyclases↗