De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

Britta Lindholm-Sethson

109.Raguse, B., Braach-Maksvytis, V., Cornell, B.A., King, L.G., Osman, P.D.J., Pace, R.J. and Wieczorek, L. (1998) Tethered lipid bilayer membranes: Formation and ionic reservoir characterisation, Langmuir 14, 648-659.

110.Kuhner, M., Tampe, R. and Sackmann, E. (1994) Lipid Monolayer and Bilayer Supported On Polymer-Films - Composite Polymer-Lipid Films On Solid Substrates, Biophys. J. 67,217-226.

11 1. Arya, A., Krull, U.J., Thompson, M. and Wong, A.E. (1985) Langmuir-Blodgett deposition of lipid films on hydrogel as a basis for biosensor development, Anal. Chim. Acta 173,331-336.

112.Bruckner-Lea, C., Petelenez, D. and Janata, J. (1990) Use of poly(octadec-1-ene-maleic anhydride) for interfacing bilayer membranes to solid supports in sensor applications, Microchim. Acta 1, 169-185.

113.Spinke, J., Yang, J., Wolf, H., Liley, M., Ringsdorf, H. and Knoll, W. (1992) Polymer-supported

bilayer on a solid substrate., Biophys. J. 63, 1667-1671.

114.Erdelen, C., HLussling, L., Naumann, R., Ringsdorf, H., Wolf, H., Yang, J., Liley, M., Spinke, J. and Knoll, W. (1994) Self-assembled disulfide-functionalised amphiphile copolymers on gold, Langmuir 10, 1246-1250.

1 15. Decher, G. (1997) Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites, Science 277, 1232-1237.

116.Lindholm-Sethson, B. (1996) Electrochemistry at ultrathin organic films at planar gold electrodes, Langmuir 12, 3305-3314.

117.Lindholm-Sethson, B., Gonzalez, J.C. and Puu, G. (1998) Electron transfer to a gold electrode from cytochrome oxidase in a lipid bilayer via a polyelectrolyte film, Langmuir 14, 6705-6708.

118.Majewski, J., Wong, J.Y., Park, C.K., Seitz, M., Israelachvili, J.N. and Smith, G.S. Structural studies of polymer-cushioned lipid bilayers, Biophys. J. 75,2363-2367, 1998.

119.Wong, J.Y., Majewski, J., Seitz, M., Park, C.K., Israelachvili, J.N. and Smith, G.S. (1999) Polymercushioned bilayers. I. A structural study of various preparation methods using neutron reflectometry, Biophys. J. 77, 1445-1457.

120.Seitz, M., Wong, J.Y., Park, C.K., Alcantar, N.A. and Israelachvili, J. (1998) Formation of tethered supported bilayers via membrane-inserting reactive lipids, Thin Solid Films 329, 767-771,

121.Gyorvary, E., Wetzer, B., Sleytr, U.B., Sinner, A,, Offenhausser, A. and Knoll, W. (1999) Lateral diffusion of lipids in silane-, dextran-, and S-layer- supported monoand bilayers, Langmuir 15, 13371347.

122.Hillebrandt, H., Wiegand, G., Tanaka, M. and Sackmann, E. (1999) High electric resistance polymer/lipid composite films on indium-tin-oxide electrodes, Langmuir, 15,845 1-8459.

123.Pagano, R.E. and Miller, I.R. (1973) Transport of Ions Across Lipid Monolayers. IV. Reduction of Polarographic Currents by Spread monolayers, J. Colloid Interface Sci., 45, 126-137.

124.Miller, I.R. (1981) Structural and energetic aspects of charge transport in lipid layers and in biological membranes, in Milazzo, G. (ed), Topics in Bioelectrochemistry and Bioenergitics, Vol. 4, John Wiley & Sons, Ltd., pp. 161-224.

125.Miller, I.R., Doll, L. and Lester, D.S. (1992) Interaction of Alamethicin, Melittin and Protein-Kinase-C With Pure and Phospholipid Monolayer Covered Mercury-Electrode Surfaces, Bioelectrochem. Bioenerg. 28, 85-103.

126.Nelson, A. and Benton, A. (1986) Phospholipid Monolayers At the Mercury Water Interface, J. Electroanal. Chem. 202,253-270.

127.Nelson, A. and Auffret, N. (1988) Phospholipid Monolayers of Di-Oleoyl Lecithin At the Mercury Water Interface, J. Electroanal. Chem. 244, 99-1 13.

128.Bizzotto, D. and Nelson, A. (1998) Continuing electrochemical studies of phospholipid monolayers of dioleyl phosphatidylcholine at the mercury - electrolyte interface, Langmuir 14, 6269-6273.

129.Nelson, A. (1991) Electrochemical Studies of Thallium(1) Transport Across Gramicidin Modified Electrode-Adsorbed Phospholipid Monolayers, J. Electroanal. Chem. 303,221-236.

130.Nelson, A. (1996) Influence of biologically active compounds on the monomolecular gramicidin channel function in phospholipid monolayers, Langmuir 12,2058-2067.

131.Nelson, A. (1997) Influence of fixed charge and polyunsaturated compounds on the monomolecular gramicidin channel function in phospholipid monolayers: Further studies, Langmuir 13, 5644-565 1.

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P. Robberecht, M. Waelbroeck and N. Moguilevsky.

In 1991, the rat secretin receptor was cloned by Ishiara et al. [4]. The receptor sequence was related to that of the calcitonin and the parathormone receptors [5,6]. The knowledge of the sequence of the secretin receptor initiated the cloning by analogy of the VIP, PACAP, glucagon, GRF, GIP, and GLP- 1 receptors, revealing the existence of a large new family of G protein coupled receptors known as the GPCR B family [7].

2. The secretin receptor

2.1. GENERAL ARCHITECTURE

The mRNA of the rat [4], rabbit [8] and human [9,10] secretin receptors, coded for proteins of 449, 445, and 440 amino acid residues respectively. Sequence analysis and modelling predicted 23 amino acid residues for a signal peptide, 120 residues for the amino terminal extra-cellular domain (N-EC), the existence of seven transmembrane domains (TM1 to TM7) connected by three extracellular (EC1 to EC3) and three intracellular loops (IC1 to IC3) and an intracellular carboxyl terminus of 50 residues (Fig. 1).

Figure 1. A secondary structure model representation of the rat secretin receptor. The putative signal peptide is bordered by a rectangle and Y indicates the glycosylable asparagine residues.

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Functional structure of the secretin receptor

Four glycosylable asparagine residues were found in the N-EC; a fifth residue was present in the EC2 domain of the rat and human sequences, but not in the rabbit one. A potential site for O-glycosylation is also present in the N-EC. Ten highly conserved Cys residues were noticed in the extracellular part of the receptor : 7 in the N-EC, 2 in EC1 and 1 in EC2 domains. In a tentative to establish the disulfide bridges, we mutated each Cys residue in serine. Six of the point-mutant receptors, C25 S, C44 S, C53 S, C67 S, C85 S, and C101 S, in the N-EC domain could not be detected by binding studies or by functional assay of adenylate cyclase activation : the mutations resulted in functional inactivation of the receptor, but it was not established if this lack of activity was due to a lack of expression at the cell surface or to a severe misfolding of an otherwise normally expressed receptor. In contrast, the four other point-mutant receptors C 1 1 S, C186 S, C193 S and C263 S, that means the first Cys residue that followed the signal peptide and the three Cys residues in the EC domains, were able to bind secretin and to be activated by secretin. However the affinity for the ligand was lower than that of the wild-type receptor. These results suggested that Cys residues 24, 44, 53, 67, 85, and 10 1 were necessary for receptor function and that two putative disulfide bridges formed by Cys residues 11, 186, 193, and 263 were functionally relevant but not essential for receptor expression. Secretin activated adenylate cyclase with a very high EC50 value (concentration required for 50 % activation of the enzyme) through the quadruple mutant C11,186,193,263 S, the four triple mutants and through double mutants C186,193 S and C186,263 S, suggesting that, in the wild-type receptor, disulfide bridges are formed between the N-EC Cys residue 11 and the first residue 186 of EC1 on one hand and between the second residue of EC1 193 and the residue 263 of EC2. Several results indicated that in the mutant receptors alternative disulfide bridges can be formed between Cys 186 and 193 or 263, suggesting that these residues are in close spatial proximity in the wild-type receptor [11].

Mutations, deletions, or exchange of parts of the secretin receptor by the corresponding domain of the parent receptors VIP, PACAP, or glucagon have been performed to delineate the areas necessary for an appropriate structure. It is however difficult to make the difference between receptors modifications leading to an impaired structure from those that modulate ligand binding without major change in the general structure. Mutations in the N-EC domain of Asp residue 49 into Arg (but not by an uncharged polar or hydrophobic residue), of the Arg residue 83 by an Asp or by a Leu residue led to almost inactive receptors. Similarly, mutations in the EC 1 domain of Asp 174 into Ala but not Asn, or mutation in the EC2 domain of Arg 255 by an Asp or a Glu residue also severely impaired ligand recognition [ 12]. No functional receptor could be obtained after replacement of N-EC or EC3 by the corresponding sequences of the glucagon receptor [13]. At the opposite, deletion of the highly charged sequence 30-33 Lys-Glu-Lys-Lys did not affect ligand recognition and receptor expression (unpublished data). Taken as a whole, these results suggest that several parts of the receptor contributed to the functional structure of the secretin receptor.

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2.2.FUNCTIONAL DOMAINS

2.2.1.Ligand binding domain

At variance with what is observed for the GPCR A group of receptors that included the bioactive amines, the small peptides, the nucleotides and the prostaglandin receptors, the N-EC domain is of paramount importance for ligand binding and discrimination among the ligands. Chow reported [14] that the exodomain alone of the secretin receptor binds secretin with a high affinity. Replacement of the secretin receptor N-EC by the VIP receptor counterpart resulted in biological responsiveness rather typical of VIP receptor. The replacement of the VIP receptor N-EC domain by the secretin

VPAC 1 receptor counterpart led to constructions that could not be studied due to a lack of expression or to a low affinity. The replacement of the sequences 13-18 and 23-29 of the secretin receptor by the VPACl receptor sequence did not change the receptor's characteristics. Introduction of the VPACl receptor sequences 103-110 or 116-120 in the secretin receptor led to constructions with a lower affinity for secretin or a higher affinity for VIP respectively [19]. Holtman et al. [15] reported the importance of the first 10 residues of the N-EC1 sequence for secretin recognition, a finding confirmed by Olde et al. [17]. It appears thus that several parts of the amino-terminal extracellular domain of the receptor contribute to the ligand recognition. By testing the functional properties of modified ligands on chimeric VPAC/secretin receptors, we obtained indirect arguments for an interaction between the carboxyl-terminal sequence 23-27, the sequence 8-15 and residue 16 of secretin with the N-EC domain of the receptor [2022]. It was also suggested that the Lys residue 15 of secretin contacts with the Asp 98 of the receptor [23]. By photo-affinity labelling of the secretin receptor with a secretin probe that had incorporated a photo-labile p-benzoyl-L-phenylalanine into position 22 and 6, it was shown by Miller's group that Leu 22 interacted with a receptor sequence comprised between amino acid 1 to 30 of the receptor and Phe 6 with Val 4 of the receptor [24,25]. The N-EC domain is not the sole domain interacting with the ligand: the His 189-Lys 190 sequence of the C-terminus of EC1 and Phe 257-Leu 258, Asn 260-Thr 261 in the amino-terminal half of EC2 also provide critical determinants, but the ligand counterpart was not established [ 18]. Several arguments suggest that the amino terminus of secretin interacted directly with the secretin receptor : a) deletion of His 1, Ser 2 and Asp 3 decreased 1000 to 10,000-fold peptide affinity and decreased the peptide efficacy : secretin (2-27) and (3-27) are partial agonists whereas secretin (4-27) is an antagonist; b) replacement of Asp 3 by Glu and Asn reduced secretin affinity 10 and 300-fold respectively [26]. We first observed [13] that the Lys residue 173 boarding TM2 and EC1 was in the vicinity of the binding region of Asp 3 as its mutation into Ileu decreased peptide affinity and makes Asn 3 and Glu 3 secretin indistinguishable. We then explored the amino acid residues surrounding that Lys

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Functional structure of the secretin receptor

residue : mutation of Asp 174 into Asn not only reduced secretin affinity but also changed receptor's selectivity as judged by a decreased secretin / Asn 3 secretin ratio [27]. When Arg 166 (in TM2) was mutated to Gln, Asp or Leu, the receptor had a very

Arg 166 is an excellent candidate for a precise contact between the ligand and the receptor : a) the positively charged residue is conserved in the whole family of receptors except the calcitonin receptor; b) receptor modelling suggests it to be rather accessible as its side chain is facing an internal pocket limited by the other TM [28]; c) according to this model, the residue is buried relatively deeply in the transmembrane domain suggesting that the ligand may enter into the receptor to change its conformation and promote the G protein activation. Analysis of the sequence of the receptor indicated that two Tyr residues located in TM1 at the same level as Arg 166 in TM2 might be involved also in the positioning of secretin amino-terminus sequence. We mutated Tyr 124 and Tyr 128 into Ala and His. Mutations of Tyr 124 reduced 10-fold the affinity of secretin and all the analogs tested. Mutation of Tyr 128 into Ala reduced 50-fold secretin affinity but also modified the capability of the receptor to discriminate the analogs modified in position 3. Ala 128 secretin receptor was unable to discriminate secretin from Glu 3 secretin. His 128 secretin receptor was almost comparable to the wild-type receptor [29].

Figure 2. Schematic representation of the possible interactions between the Asp 3 residue ofsecretin and the lateral chains of the amino acids of TMI and TM2.

Taken together, the analysis of the receptors mutated in TM1 and TM2 supported the hypothesis that secretin Asp 3 (or Glu) residues are anchored by ionic interactions with the Lys 173 and Arg 166 residues while the Asp 3 (or Asn) residues may form hydrogen bonds with Tyr 128. Replacement of the two basic residues by uncharged residues decreased markedly the affinity for secretin and Glu 3 secretin but not for the uncharged Asn 3 secretin analog. In contrast, replacement of Tyr 128 by an Ala residue decreased the receptor affinity for secretin and the isosteric Asn 3 secretin analog, but

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not for the Glu 3 secretin. Replacement of Tyr 128 by His which may also form hydrogen bonds had little consequence on the recognition profile. A model for the positioning of the Asp 3 residue in the receptor is presented in Fig. 2.

2.2.2. Coupling of the receptor to the G protein.

It was shown that secretinbut not secretin analogs in which a Tyr residue was introduced for a Leu residue in position 10 and 13, stimulated adenylate cyclase activity at low concentrations and phosphatidylinositol bisphosphate hydrolysis at higher concentrations [30]. The dual activation of the cyclic AMP and the inositol trisphosphate / Ca++ pathways, that was reported for other parents receptors [31,321 was not described for the wild-type recombinant secretin receptor expressed in CHO cells (may be it was not yet tested). So far, the preferential, if not the sole, coupling

mechanism of the secretin receptor is Gas and adenylate cyclase activation. There is to our knowledge no study investigating the precise domain of the receptor responsible for

that coupling. Mutation of His 156 into Arg (in TM2 near the inner face of the membrane) or Thr 322 into Pro (in TM6 near the inner face of the membrane) resulted in a constitutively active receptor, that means that the unstimulated receptor may couple to adenylate cyclase. Interestingly, the double mutant Arg 156 / Pro 322 had a greater activity than each individual mutant and secretin behaves like an inverse agonist, that means it reduced the basal adenylate cyclase activity [33]. These data suggested that the coupling to adenylate cyclase through interaction with Gas involved a large area at the inner face of the membrane.

2.2.3. Desensitisation ofthe receptor.

As mostif not allthe G protein coupled receptors, the secretin receptor desensitises rapidly and is internalised through a not yet identified mechanism, insensitive to sucrose but sensitive to Con A [34]. After secretin exposure, the secretin receptor expressed in Cos or CHO cells is rapidly phosphorylated, mainly on Thr residues located essentially in the carboxyl-terminal intracellular tail [35]. Truncation of this part of the receptor does not alter the ligand recognition, nor the coupling to adenylate cyclase but prevents its phosphorylation [36]. However, the truncated receptor remains still internalisable. Thus a dual mechanism limits secretin action : a rapid phosphorylation decreases the cyclic AMP production and an internalisation independent of phosphorylation occurs. In transfected HEK 293 cells, it was demonstrated that protein kinases A and C do not markedly affect receptor phosphorylation that is mainly induced by the GRK2 and GRK5 kinases [37].

3. Conclusions and perspectives

Secretin is mainly a hormone controlling the pancreatic fluid secretion through interaction with acinar and duct pancreatic cells [3] and the bile flow through

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interaction with cholangiocytes [3 8]. Intravenous injections of secretin were used for years to evaluate the exocrine pancreatic function in human. The natural porcine secretin used first was contaminated by the neurohormone cholecystokininpancreozymin (CCK-PZ) that contracts vigorously the gallbladder; since the availability of essentially pure porcine secretin obtained from the Karolinska Institute, and later on from Kabi-Vitrum, the effect of secretin and CCK-PZ have been dissociated. The functional secretin-cholecystokinin test was performed by measuring fluid and enzyme secretion in duodenal aspirates. This was a good clinical test when performed by trained physicians. Its interest dropped however when retrograde cholangio-wirsungography was introduced as a routine endoscopic procedure : the visualisation of the pancreatic and the biliary tract was more helpful for the clinician than the functional tests. Furthermore, echography became the best non-invasive technique for gallbladder volume and contractility evaluation. Catheterisation of the main pancreatic duct with a balloon catheter allowed the analysis of pure pancreatic juice in response to secretin. This technique was however difficult to perform and was considered as an invasive one limited to experimental studies. More recently, the imaging of the pancreatic tree was possible by magnetic resonance cholangiopancreatography after secretin injection. This non-invasive technique will probably be in a near future the reference for pancreatic exploration. The systematic injection of secretin to patients suffering of gastrointestinal tract diseases will hopefully led to the discovery of secretin-linked pathologies (hypersecretory responses that could be due to constitutively active secretin receptors, absence of secretin response ...).

Secretin, or preferably stable analogues - peptidic or non peptidic - could be of a great therapeutic value : repeated administration of secretin by inducing a high pancreatic flow rate might reduce the formation of pancreatic stones; secretin administered after fragmentation of pancreatic calculi by ultrasound bombardment will efficiently eliminate the stone fragments.

Secretin is also a neuropeptide regulating tyrosine hydroxylase activity in PC 12 cells and in sympathetic ganglia [39]. It activates also adenylate cyclase in striatal neurons from embryonic mouse brain grown in cultures [40]. Its effect on adult rat brain occurs however at high concentrations only [41]. Recently, case reports of dramatic improvements of children suffering from autism were reported after secretin administration [42]. However, a recent double-blind study concluded that a single dose of human secretin was not an effective treatment for autism [43]. The use of more stable analogs with a facilitated penetration into the brain should be evaluated.

How may the knowledge of the interactions between ligand and receptor contribute to the conception of peptidominetic ligands of potential therapeutic use ? Actually, most - if not all - peptidominetics have been discovered by chance (for instance the antibiotic erythromycin mimics the effect of the hormone Motilin by interacting with the motilin receptor) or by a systematic screening of large chemical libraries. This approach requires only an appropriate cellular model expressing the receptor of interest coupled to an easily detected effector system and the access to a large number of molecules. Another approach, based on the knowledge of the binding domain of the receptor is intellectually more rewarding but difficult : it requires a feed-back

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