SRIF receptors are membrane-bound
glycoproteins. To structurally identify the
carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to
lectin affinity chromatography. Solubilized SRIF receptors specifically bound to
wheat germ agglutinin-
lectin affinity columns but not to
succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L.
lectin affinity column suggested that
sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to
concanavalin A, Dolichus biflorus
agglutinin, Ulex europeaus I, and
Jacalin lectin affinity columns suggests that high
mannose, N-
acetylgalactosamine,
fucose, and O-linked
carbohydrates are not associated with receptor. To investigate the functional role of the
carbohydrate groups in brain SRIF receptors, specific
sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]
MK 678 to SRIF receptors was determined. Treatment of the receptor with
endoglycosidase D did not affect the specific binding of [125I]
MK 678 to the solubilized SRIF receptors, consistent with the finding from
lectin affinity chromatography that high
mannose-type
carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with
peptide-N-glycosidase F and
endoglycosidases H and F reduced [125I]
MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked
carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with
neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with
neuraminidase from Newcastle disease virus did not affect [125I]
MK 678 binding to the receptor. These findings suggest that
sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the
pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with
neuraminidase (V.
cholera) greatly reduces high affinity [125I]
MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit
forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to
GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with
neuraminidase from V.
cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]
MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)