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7-Bromoacetyl-7-desacetylforskolin (BrAcFsk), an alkylating derivative of forskolin, activated adenylyl cyclase and irreversibly blocked high affinity forskolin binding sites in human platelet membranes and rat brain membranes (Laurenza et al., 1990). Photoincorporation of an iodinated arylazido derivative of forskolin, 125I-6-AIPP-Fsk, into adenylyl cyclase in bovine brain membranes was irreversibly inhibited by BrAcFsk but not by 1,9-dideoxy-BrAcFsk, suggesting that BrAcFsk was reacting specifically with a nucleophilic group(s) at the forskolin binding site of adenylyl cyclase. Immunoblotting with antiforskolin antiserum demonstrated that partially purified bovine brain adenylyl cyclase had incorporated BrAcFsk. The interaction of BrAcFsk with the glucose transporter in human erythrocyte membranes was examined in a similar manner. Photoincorporation of 125I-7-AIPP-Fsk, an iodinated arylazido derivative of forskolin which is specific for the glucose transporter, into the glucose transporter was not irreversibly inhibited by BrAcFsk, suggesting that, in contrast to adenylyl cyclase, there is no reactive nucleophilic group at the forskolin binding site on the human erythrocyte glucose transporter. The immunoblotting procedure with antiforskolin antiserum confirmed that BrAcFsk was not covalently attached to human erythrocyte glucose transporter.

Aminoalkylcarbamate derivatives of forskolin have been synthesized at the 6- and 7-hydroxyl positions which have different selectivity for adenylyl cyclase and a glucose transporter, respectively. They were radioiodinated using the Bolton-Hunter reagent to yield [125I]-2-[3-(4-hydroxy-3-iodophenyl)propanamido]-N-ethyl-6- (aminocarbonyl)forskolin ([125I]6-IHPP-Fsk) and [125I]-2-[3-(4-hydroxy-3-iodophenyl)(propanamidol]-N-ethyl-7- (aminocarbonyl)-7-desacetylforskolin ([125I]7-IHPP-Fsk) and tested as autoradiographic probes for adenylyl cyclase and a glucose transporter. In slide-mounted rat brain sections [125I]6-IHPP-Fsk binding was potently inhibited by 1 microM 6-HPP-Fsk (95%) but unaffected by 500 mM D-glucose. In contrast, [125I]7-IHPP-Fsk was only partially inhibited by 1 microM 6-HPP-Fsk (37%), but residual [125I]7-IHPP-Fsk binding was further inhibited 56% by 500 mM D-glucose. These data suggest that while [125I]6-IHPP-Fsk binds exclusively to adenylyl cyclase, a significant fraction of [125I]7-IHPP-Fsk is binding to a glucose transporter in brain. Autoradiographic patterns of [125I]6-IHPP-Fsk and glucose-sensitive [125I]7-IHPP-Fsk binding were different. [125I]6-IHPP-Fsk binding was heterogeneously distributed and resembled [3H] forskolin binding. Highest densities of binding sites were noted in olfactory tubercle, caudate putamen, nucleus accumbens, pyramidal and granule cell layers of hippocampus, molecular layer of cerebellum and substantia nigra. In contrast, of glucose-sensitive [125I]7-IHPP-Fsk, binding appeared more homogeneous and similar to [3H]cytochalasin B, a compound which inhibits glucose transport. Highest densities of binding were noted in caudate putamen, nucleus accumbens, cerebral cortex and molecular layer of cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Two radioiodinated derivatives of forskolin, [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk, were synthesized as specific ligands for adenylyl cyclase and glucose transporter, respectively. [125I]6-IHPP-Fsk bound to bovine brain homogenates with a Kd of 9 nM and binding was inhibited by forskolin but not 1,9-dideoxyforskolin, cytochalasin B, or D-glucose. [125I]7-IHPP-Fsk bound to bovine brain homogenates at two classes of binding sites with Kd's of 56 nM and 4.7 microM; cytochalasin B and D-glucose inhibited 75% of the high affinity binding while having no effect on the low affinity binding. [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk were used to localize adenylyl cyclase and glucose transporter in rat brain by receptor autoradiography. The pattern of binding obtained with [125I]6-IHPP-Fsk was similar to that observed using [3H]forskolin to detect adenylyl cyclase. In contrast, the pattern of binding obtained with [125I]7-IHPP-Fsk was similar to that observed by others using [3H]cytochalasin B to detect glucose transporter. These iodinated ligands are selective for adenylyl cyclase and glucose transporter and require significantly shorter exposure times to yield autoradiographs than tritiated ligands.

(Aminoalkyl)carbamates of forskolin were synthesized at the 6- and 7-hydroxyl positions of forskolin with the length of the alkyl chain varying from ethyl to heptyl. Two of these derivatives, 7-[[(2-aminoethyl)amino]carbonyl]-7-desacetylforskolin (2) and 6-[[(2-aminoethyl)amino]carbonyl]forskolin (3), were used to synthesize iodinated derivatives of forskolin that bind with high affinity to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes, respectively. Hydroxyphenyl derivatives of forskolin were prepared from the (aminoalkyl)carbamates and tested for their ability to bind to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes. The 6-derivative (18) of forskolin had a Kd of 9 nM at adenylyl cyclase and was more potent than either the 7-derivatives or the 6-derivatives of 7-desacetylforskolin. The 7-derivatives were more potent at binding to the glucose transporter than forskolin. In contrast, the 6-derivatives had Kd's greater than 100 microM at the glucose transporter. Isothiocyanates and N-bromoacetyl derivatives were synthesized from 2 and 3 as potential alkylating agents for forskolin binding sites. The alkylating agents produced an irreversible loss of forskolin binding to adenylyl cyclase. In contrast, the alkylating agents bound reversibly to the glucose transporter.

Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.