Thyrotropin-releasing hormone-diphtheria toxin-related polypeptide conjugates. Potential role of the hydrophobic domain in toxin entry.

We have separately coupled a modified thyrotropin-releasing hormone (TRH) molecule to two diphtheria toxin-related polypeptides, CRM26 and CRM45. Both polypeptides are enzymatically active but only CRM45 contains the hydrophobic domain of the toxin molecule. The TRH-CRM45 conjugate caused a 50% inhibition of protein synthesis in GH3 rat pituitary cells at 3 X 10(-9) M. CRM45 alone was 200 to 500 times less toxic than the conjugate. Intoxication by TRH-CRM45 was prevented by excess TRH, preincubation with diphtheria antitoxin, or reduction of the disulfide cross-link. In addition, TRH-CRM45 was no more toxic than CRM45 itself to 3T3 cells which lack TRH receptors. In contrast, TRH-CRM26, although it retained enzymatic activity, was nontoxic for GH3 cells even at 10(-7) M. Binding experiments showed that both conjugates compete for TRH receptors with comparable affinities. The potential role of the hydrophobic domain in toxin entry is discussed.