Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

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 EC 50 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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