Structural analysis of a distinct subtype of CCK receptor on human gastric smooth muscle tumors.
Human gastric smooth muscle tumors (leiomyosarcomas) have been shown to express cholecystokinin (CCK) binding sites that are functionally similar to physiologically important receptors present on their cells of origin. In this work, we have applied affinity-labeling techniques using 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) to attempt to define the ligand-binding subunit of this receptor, and we have used the receptor antagonist L364,718 and deglycosylating enzymes to compare this molecule with well-defined CCK receptors on the classical peripheral targets (pancreas and gallbladder) of this hormone. To validate the use of 125I-D-Tyr-Gly-[Nle28,31]CCK-(26-33) for this tissue, we demonstrated that it bound to leiomyosarcoma membranes in a rapid, reversible, saturable, specific, and high-affinity manner (Kd = 0.8 nM). Although previous affinity labeling of this tissue with a CCK-33-based probe identified multiple bands, only one of those candidate proteins was predominantly labeled in the present work (Mr 100,000) by using a probe that is cross-linked through a site in greater proximity to the receptor-binding domain. Labeling was inhibited in a concentration-dependent manner by CCK-8 but not by structurally unrelated ligands. Although endo-beta-N-acetyl-glucosaminidase F digestion shifted this band by Mr 5,000, demonstrating that it was a glycoprotein, the deglycosylation product was very different from other CCK receptors studied. Also, unlike pancreatic and gallbladder CCK receptors, affinity labeling of this receptor was not affected by L364,718. These observations confirm that the gastric smooth muscle tumor CCK receptor represents a receptor subtype that is distinct from other peripheral CCK receptors, biochemically as well as functionally.
Pearson
R K
RK
Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Hadac
E M
EM
Miller
L J
LJ
D000328:Adult; D000368:Aged; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D005753:Gastric Mucosa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007890:Leiomyosarcoma / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D011949:Receptors, Cholecystokinin / Q000302:isolation & purification / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000378:metabolism*
D011949:Receptors, Cholecystokinin